Interaction of tobacco-specific toxicants with the neuronal α7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders

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Abstract

Pulmonary neuroendocrine cells function as hypoxia-sensitive chemoreceptors, and they release peptides and biogenic amines that are important mediators of pulmonary neonatal adaptation. Some of these products additionally act as autocrine growth factors. Increased numbers of pulmonary neuroendocrine cells have been observed in several smoking-associated pediatric lung disorders such as bronchopulmonary dysplasia, cystic fibrosis, sudden infant death syndrome, and asthma. Disturbed pulmonary neuroendocrine function has been implicated in the etiology of this disease complex. One of the most common smoking-associated lung cancer types, small cell lung carcinoma, expresses phenotypic and functional features of pulmonary neuroendocrine cells. We, as well as others, have shown that the release of the autocrine growth factors 5-hydroxytryptamine (5-HT, serotonin) and mammalian bombesisn/gastrin releasing peptide (MB/GRP) by cell lines derived from human small cell lung carcinoma or fetal hamster pulmonary neuroendocrine cells are regulated by a neuronal nicotinic acetylcholine receptor comprised of α7 subunits. In radio-receptor assays, nicotine and the nicotine-derived carcinogenic nitrosamines N′-nictrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bound with high affinity to this receptor with affinitites NNK>nicotine>NNN. Binding of nicotine or NNK to the α7 receptor resulted in calcium influx and overexpression and activation of the serine–threonine protein kinase Raf-1. In turn, this event lead to overexpression and activation of the mitogen activated (MAP) kinases extracellular signal regulated kinase 1 (ERK1) and extracellular signal regulated kinase 2 (ERK2) and stimulation of DNA synthesis accompanied by an increase in cell numbers in fetal pulmonary neuroendocrine cells and small cell carcinoma cells. Exposure of fetal pulmonary neuroendocrine cells for 6 days to NNK caused a prominant up-regulation of Raf-1. Our findings suggest that chronic exposure to nicotine and NNK in pregnant women who smoke may up-regulate the α7 nicotinic receptor as well as components of its associated mitogenic signal transduction pathway, thus increasing the susceptibilities of the infants for the development of pediatric lung disorders. Similarly, up-regulation of one or several components of this nicotinic receptor pathway in smokers may be an important factor for the development of small cell lung carcinoma.

Introduction

This manuscript represents a review of our work during the past 3 years and includes new, unpublished data, as well as some data which have been previously published in a different context. Appropriate reference is given in the text and figure legends.

Pulmonary neuroendocrine cells are specialized lung cells which co-express features of neurons and endocrine cells (Becker, 1984). These cells are abundant in the neonate, and decrease rapidly in number by 12 months of age Cutz, 1997, Cutz et al., 1985. Pulmonary neuroendocrine cells are hypoxia-sensitive chemoreceptors and are thought to be important mediators of pulmonary neonatal adaptation, particularly the onset of breathing Cutz, 1997, Cutz et al., 1985. Pulmonary neuroendocrine cells demonstrate hyperplasia accompanied by elevated levels of their secretory products in a variety of pediatric pulmonary diseases, such as bronchopulmonary dysplasia (Johnson and Georgieff, 1989, Johnson et al., 1982, Cutz, 1997, cystic fibrosis Johnson et al., 1988, Cutz, 1997, sudden infant death syndrome Cutz et al., 1988, Cutz et al., 1996, Johnson and Georgieff, 1989, and asthma Stanislawski et al., 1981, Johnson and Georgieff, 1989, Aguayo, 1993. Functional abnormalities of these cells may contribute to the pathophysiology of this family of pediatric lung diseases because their peptide and biogenic amine secretory products modulate the tonus of bronchial and vascular smooth muscle, modulate neurotransmission, and stimulate the growth of epithelial lung cells and fibroblasts Willey et al., 1984, Johnson et al., 1988, Seuwen et al., 1988, Sunday, 1988, Schuller and Hegedus, 1989, Rozengurth and Sinneth-Smith, 1990, Cutz, 1997. The most prominent product of pulmonary neuroendocrine cells for which effects on cell growth, smooth muscle tension and neuromodulation have been documented, is the biogenic amine 5-hydroxytryptamine (Cutz, 1997; 5-HT, serotonin). Research into the role of 5-HT in pediatric pulmonary disease has focused on its well-documented constricting effects on bronchial and vascular smooth muscle Johnson et al., 1988, Lechin et al., 1996, Cutz, 1997. Although it has been known for some time that 5-HT stimulates the growth of fibroblasts, its growth stimulating effects on pulmonary neuroendocrine cells and small cell carcinima has been only recently discovered Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998.

Epidemiologic studies have established that babies of mothers who smoke during pregnancy are at increased risk to develop sudden infant death syndrome Cutz et al., 1988, Johnson and Georgieff, 1989, Malloy et al., 1992, Schoendorf and Kiely, 1992, Schellscheidt et al., 1997 and asthma Hu et al., 1997, Oliveti et al., 1996. Moreover, both of these diseases as well as bronchopulmonary dysplasia and cystic fibrosis are exacerbated in newborns and infants exposed to second-hand smoke Aguayo, 1993, Cutz et al., 1996, Knight et al., 1998. However, the underlying mechanisms of these effects are far from understood. Nicotine is a well-documented secretagogue for pulmonary neuroendocrine cells and causes exocytosis of their dense-cored cytoplasmic granules which are the storage site of peptides and serotonin Lauweryns et al., 1977, Tabbasian et al., 1989, Cutz, 1997. Similarly, nicotine stimulated the release of 5-HT from human small cell lung carcinoma cells in culture Codignola et al., 1994, Schuller and Orloff, 1998. As it is well established that the biological effects of nicotine are mediated by binding to nicotinic acetylcholine receptors, these findings suggest that the release of 5-HT is under autonomic control via members of the nicotinic acetylcholine receptor family.

Smoking has long been recognized as the single most important risk factor for the development of lung cancer (Weiss, 1983; Cook et al., 1993). While non-small cell lung carcinomas develop also in a significant number of non-smokers, small cell lung carcinoma is extremely rare in non-smokers (Weiss, 1983; Cook et al., 1993). Chronic non-neoplastic pulmonary disease (bronchitis/bronchiolitis, emphysema, chronic obstructive pulmonary disease, asthma) has more recently emerged as a risk factor for lung cancer in both smokers and non-smokers Osann, 1991, Weiss, 1991, Park et al., 1995. In fact, a recent study has shown that elevated CO2 levels comparable to those in the diseased lung stimulate the release of 5-HT, resulting in the activation of the MAP kinase cascade in SCLC (Merryman et al., 1997). Small cell lung carcinoma expresses phenotypic and functional characteristics of pulmonary neuroendocrine cells (Gazdar and Carney, 1984). All pulmonary neuroendocrine cells and small cell lung carcinoma cells have the ability to synthesize and release 5-HT (Becker, 1984; Gazdar, 1984), suggesting an important function of this biogenic amine in this histologic lung cancer type. This interpretation gains strong support from the recent finding that 5-HT is an autocrine growth factor for this cancer type as well as for pulmonary neuroendocrine cells Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998, Codignola et al., 1994.

The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are formed from nicotine by nitrosation (Fig. 1) during the curing of tobacco and in the mammalian organism Fischer et al., 1990, Hecht and Hoffmann, 1990. NNK is a potent lung carcinogen in all animal species tested, and is thought to contribute significantly to the high lung cancer burden associated with smoking (Hecht and Hoffmann, 1990). On the other hand, NNN, which more closely resembles nicotine in structure than NNK (Fig. 1) is a weak carcinogen in laboratory animals. It is well established that reactive metabolites of NNK or NNN may form DNA-methylating and pyridyloxobutylating products (Fig. 2), resulting in activating point mutations of the Ki-ras gene Belinsky et al., 1988, Hecht et al., 1993a, Hecht et al., 1993b. With respect to the central role of ras as a signal transducer involved in mitogenic pathways of mammalian cells (Lange-Carter and Johnson, 1994), this event is thought to be primarily responsible for the development of lung tumors in response to NNK Belinsky et al., 1988, Hecht et al., 1993a, Hecht et al., 1993b. However, point mutations of the ras gene have only been identified in non-small cell lung carcinomas whereas they are consistently absent in small cell lung carcinoma Mitsudomi et al., 1991, Wagner et al., 1993. The α7 nicotinic acetylcholine receptor and its associated mitogenic signal trasnduction pathway is emerging as an important growth regulator of pulmonary neuroendocrine cells and small cell lung carcinoma Schuller, 1989, Schuller and Hegedus, 1989, Schuller and Orloff, 1998, Cattaneo et al., 1993, Cattaneo et al., 1997, Codignola et al., 1994 and may be critically involved in the development of neoplastic and non-neoplastic pulmonary diseases.

Section snippets

Tissue culture

Cultures of fetal pulmonary neuroendocrine cells were established from fetal hamster lung periphery harvested on day 15 of gestation as previously described Linnoila et al., 1993, Schuller, 1994. Following disaggregation of the tissue (100 mg) with 3 ml trypsin/EDTA for 60 min at 37°C, complete RPMI medium was added to yield 106 cells/ml, and 2 ml each of the resulting cell suspension was used to seed 6 T75 tissue culture flasks. The cultured cells were enriched to >80% of pulmonary

Results

RT-PCR assays with the primer for the human α7 nicotinic acetylcholine receptor revealed mRNA of this receptor in fetal hamster pulmonary neuroendocrine cells and human small cell lung carcinoma cells Fig. 3, Fig. 4. Sequence comparison of the 611 bp between the primers of the RT-PCR fragment from fetal hamster pulmonary neuroendocrine cells with the comparable sequence of human α7 mRNA by a maximum matching program of DNASIS showed a matching percentage of 89%. A Blast search of Genbank using

Discussion

The data presented in this review suggest that pediatric smoking-associated pulmonary diseases and small cell lung carcinoma may be caused by the direct chronic stimulation of an α7 nicotinic acetylcholine receptor-initiated autocrine loop by nicotine or NNK. While it is well established that the Raf-1/MAP kinase cascade is a major mitogenic signal transduction pathway expressed in human small cell lung carcinoma Bunn et al., 1993, Heysley and Johnson, 1998, the expression and function of this

Acknowledgements

This study is supported by Public Health Service grant CA51211 with the National Cancer Institute and a grant with the VERUM Foundation (Munich, Germany).

References (73)

  • H.M. Schuller

    Cell type-specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines

    Biochem. Pharmacol.

    (1989)
  • H.M. Schuller et al.

    Effects of endogenous and tobacco-related amines and nitrosamines on cell growth and morphology of a cell line derived from a human neuroendocrine lung cancer

    Toxicol. In Vitro

    (1989)
  • H.M. Schuller et al.

    Tobacco-specific nitroamines: ligands for nicotinic acetylcholine receptors in human lung cancer cell lines

    Biochem. Pharmacol.

    (1998)
  • M.E. Williams et al.

    Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium channel subtype

    Neuron

    (1992)
  • S.M. Aguayo

    Pulmonary neuroendocrine cells in tobacco-related lung disorders

    Anat. Rec.

    (1993)
  • E.L. Barry et al.

    Expression and antibody inhibition of P-type calcium channels in human small cell lung carcinoma cells

    J. Neurosci.

    (1995)
  • K.L. Becker

    The endocrine lung

  • S.A. Belinsky et al.

    Activation of the K-ras protooncogene in lung tumors of mice treated with 4-(methylnitrsamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosodimethylamine (NDMA)

    Cancer Res.

    (1988)
  • C.R. Breese et al.

    Effect of smoking history on [3H]-nicotine binding in human postmortem brain

    J. Pharmacol. Exp. Ther.

    (1997)
  • P.A. Bunn et al.

    Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity

    Cancer Res.

    (1993)
  • M.G.A. Cattaneo et al.

    Nicotine stimulates a serotonergic autocrine loop in human small cell carcinoma

    Cancer Res.

    (1993)
  • M.G. Cattaneo et al.

    Mechanisms of micotgen-activated protein kinase activation by nicotine in small cell lung carcinoma cells

    Biochem. J.

    (1997)
  • R.L. Cook et al.

    Small cell lung cancer: etiology, biology, clinical features, staging and treatment

    Curr. Probl. Cancer

    (1993)
  • E. Cutz

    Cellular and Molecular Biology of Airway Chemoreceptors

    (1997)
  • E. Cutz et al.

    Pulmonary neuroendocrine cells in SIDS. An immunohistochemical and quantitative study

    Ann. NY Acad. Sci.

    (1988)
  • E. Cutz et al.

    Neuroendocrine cells in the developing human lung — morphologic and functional considerations

    Ped. Pulmonol.

    (1985)
  • E. Cutz et al.

    Maternal smoking and pulmonary neuroendocrine cells in sudden infant death syndrome

    Pediatrics

    (1996)
  • R.E. Farrell

    RNA Methodologies

    (1993)
  • R.E. Farrell

    DNA amplification

    Immunol. Invest.

    (1997)
  • M. Finnegan et al.

    Investigation of the expression of housekeeping genes in non-Hodgekin's lymphoma

    Leuk. Lymphoma

    (1993)
  • S. Fischer et al.

    Investigation on the origin of tobacco-specific nitrosamines in mainstream smoke of cigarettes

    Carcinogenesis

    (1990)
  • A.F. Gazdar et al.

    Endocrine properties of small cell carcinoma of the lung

  • S.S. Hecht et al.

    Tobacco-specific nitrosamine adducts. Studies in laboratory animals and humans

    Environ. Health Perspect.

    (1993)
  • S.S. Hecht et al.

    Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites

    Carcinogenesis

    (1993)
  • S.S. Hecht et al.

    Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco-smoke

    Carcinogenesis

    (1990)
  • Heysley, L.E., Johnson, G.L., 1998. Signal transduction abnormalities in lung cancer. In: Kane, M.A., Bunn, P.A. Jr....
  • Cited by (0)

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