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The nicotinic CHRNA5/A3/B4 cluster: dual role in nicotine addiction and

Ma Reina D. Improgo University of Massachusetts Medical School

Et al.

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Repository Citation Improgo MD, Scofield MD, Tapper AR, Gardner PD. (2010). The nicotinic acetylcholine receptor CHRNA5/ A3/B4 gene cluster: dual role in nicotine addiction and lung cancer. GSBS Student Publications. https://doi.org/10.1016/j.pneurobio.2010.05.003. Retrieved from https://escholarship.umassmed.edu/ gsbs_sp/1682

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Progress in Neurobiology

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The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: Dual role in nicotine addiction and lung cancer

Ma. Reina D. Improgo, Michael D. Scofield, Andrew R. Tapper, Paul D. Gardner *

Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, 303 Belmont Street, Worcester, MA 01604, United States

ARTICLE INFO ABSTRACT

Article history: More than 1 billion people around the world smoke, with 10 million cigarettes sold every minute. Received 30 March 2010 Cigarettes contain thousands of harmful chemicals including the psychoactive compound, nicotine. Received in revised form 15 May 2010 Nicotine addiction is initiated by the binding of nicotine to nicotinic acetylcholine receptors, ligand- Accepted 27 May 2010 gated cation channels activated by the endogenous neurotransmitter, acetylcholine. These receptors serve as prototypes for all ligand-gated ion channels and have been extensively studied in an attempt to Keywords: elucidate their role in nicotine addiction. Many of these studies have focused on heteromeric nicotinic Nicotinic acetylcholine receptors acetylcholine receptors containing a4 and b2 subunits and homomeric nicotinic acetylcholine receptors CHRNA5/A3/B4 gene cluster containing the a7 subunit, two of the most abundant subtypes expressed in the brain. Recently however, Nicotine addiction Lung cancer a series of linkage analyses, candidate-gene analyses and genome-wide association studies have brought Genome-wide association studies attention to three other members of the nicotinic acetylcholine receptor family: the a5, a3 and b4 subunits. The encoding these subunits lie in a genomic cluster that contains variants associated with increased risk for several diseases including nicotine dependence and lung cancer. The underlying mechanisms for these associations have not yet been elucidated but decades of research on the nicotinic receptor gene family as well as emerging data provide insight on how these receptors may function in pathological states. Here, we review this body of work, focusing on the clustered nicotinic acetylcholine receptor genes and evaluating their role in nicotine addiction and lung cancer. ß 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 213 2. Neuronal nicotinic acetylcholine receptors...... 213 2.1. General overview ...... 213 2.2. Structural and functional properties of nAChRs ...... 213 2.3. The CHRNA5/A3/B4 gene cluster ...... 215 3. Regulation of CHRNA5/A3/B4 expression ...... 215 3.1. Expression patterns of the CHRNA5/A3/B4 genes ...... 215 3.1.1. CHRNA3 ...... 215 3.1.2. CHRNA5 ...... 215 3.1.3. CHRNB4 ...... 217 3.2. Transcriptional regulation of the CHRNA5/A3/B4 genes ...... 217 3.2.1. CHRNA3 ...... 217 3.2.2. CHRNA5 ...... 218 3.2.3. CHRNB4 ...... 218 4. Role in pathological states...... 218 4.1. Nicotine addiction...... 218

Abbreviations: ACh, acetylcholine; ASCL1, achaete–scute complex homolog-1; CNS, central nervous system; ChIP, chromatin immunoprecipitation; CNR4, conserved noncoding region 4; DA, dopamine; DHbE, dihydro-berythroidine; GABA, g-amino butyric acid; GWAS, genome-wide association study; hnRNP K, heterogeneous nuclear ribonucleoprotein K; KO, knockout; MLA, methyllycaconitine; nAChR, nicotinic acetylcholine receptor; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-butanone; NNN, N- nitrosonornicotine; NSCLC, non-small cell lung carcinoma; PNS, peripheral nervous system; SNP, single nucleotide polymorphism; SCLC, small cell lung carcinoma; SCG, superior cervical ganglion; VTA, ventral tegmental area; WT, wildtype. * Corresponding author. Tel.: +1 508 856 4035; fax: +1 508 856 4130. E-mail address: [email protected] (P.D. Gardner).

0301-0082/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2010.05.003 M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 213

4.2. Lung cancer ...... 220 4.3. Other diseases...... 220 5. Conclusions ...... 221 Acknowledgements ...... 221 References...... 221

1. Introduction non-neuronal cells such as lung, glia, keratinocytes, endothelial cells, as well as cells of the digestive and immune systems, and The molecular cloning of nicotinic acetylcholine receptors evidence is accumulating indicating that the receptors play crucial (nAChRs) from brain cDNA libraries in the mid-1980s was a roles in signal transduction underlying many physiological watershed event as it opened the window to not only a molecular processes outside the nervous system (Arredondo et al., 2001; understanding of cholinergic signaling within the nervous system, Battaglioli et al., 1998; Gahring et al., 2004b; Gahring and Rogers, but also to a structural understanding of how members of the Cys- 2006; Kawashima and Fujii, 2003; Macklin et al., 1998; Maus et al., loop superfamily of ligand-gated ion channels function. In addition 1998; Nguyen et al., 2000; Spindel, 2003; Wang et al., 2001; to neuronal and muscle nAChRs, members of this family include Wessler and Kirkpatrick, 2008). The importance of nAChR- the ionotropic receptors for glycine, 5-hydroxytryptamine (5-HT3), mediated signaling is reflected in the many pathologies in which and g-aminobutyric acid (GABAA and GABAC)(Dani and Bertrand, cholinergic signal transduction is compromised. For example, 2007; Le Nove`re and Changeux, 1995). As the cloning frenzy significant alterations in nAChR expression and function have been subsided, a tremendous amount of effort was put forth to documented in several diseases such as Alzheimer’s disease, understand the pharmacological and biophysical diversity of autosomal dominant nocturnal frontal lobe epilepsy, megacystis- nAChRs. As a result, nAChRs are among the most well understood microcolon-intestinal hypoperistalsis syndrome, Parkinson’s dis- allosteric membrane from a structural and functional ease, schizophrenia, and Tourette’s disease (De Fusco et al., 2000; point of view (Albuquerque et al., 2009). Furthermore, nAChRs Isacson et al., 2002; Lena and Changeux, 1997; Perl et al., 2003; have emerged as key therapeutic targets for a variety of Perry et al., 2001; Quik, 2004; Quik et al., 2007; Richardson et al., pathologies including schizophrenia, depression, attention deficit 2001; Silver et al., 2001; Steinlein et al., 1995; Teaktong et al., hyperactivity disorder, Alzheimer’s disease and of course, tobacco 2003; Whitehouse et al., 1988; Zanardi et al., 2002). In addition, addiction (Arneric et al., 2007; Levin and Rezvani, 2007; Romanelli nAChRs are key players in the initial steps and subsequent et al., 2007; Taly et al., 2009). More recently, genetic studies have downstream health consequences of nicotine addiction (Kedmi identified single nucleotide polymorphisms (SNPs) in the chromo- et al., 2004; Laviolette and van der Kooy, 2004). Coupled to this is a somal locus encoding three nAChR genes as risk factors for (1) growing awareness that nAChRs may directly contribute to the nicotine dependence (2) lung cancer, (3) chronic obstructive pathogenesis of lung cancer (Catassi et al., 2008; Egleton et al., pulmonary disease, (4) alcoholism and (5) peripheral arterial 2008; Schuller, 2008, 2009; Song et al., 2008). disease (Amos et al., 2008; Berrettini et al., 2008; Bierut et al., 2008; Caporaso et al., 2009; Freathy et al., 2009; Hung et al., 2008; Pillai 2.2. Structural and functional properties of nAChRs et al., 2009; Saccone et al., 2009b, 2007; Sasaki et al., 2009; Schlaepfer et al., 2008; Stevens et al., 2008; Thorgeirsson et al., Neuronal nAChRs are transmembrane proteins that form 2008; Weiss et al., 2008). This cluster of nAChR genes encodes the pentameric structures assembled from a family of subunits that a3, a5 and b4 subunits (designated CHRNA5/A3/B4) (Boulter et al., include a2–a10 and b2–b4(Brejc et al., 2001; Cooper et al., 1991). 1990). As a result of these genetic studies, new attention has been Each nAChR subunit consists of an approximately 200-residue brought to bear on the CHRNA5/A3/B4 genes and their encoded extracellular N-terminus, four transmembrane segments (desig- subunits. This review summarizes recent work on the clustered nated M1–M4), a variable intracellular loop (100–200 residues) nAChR subunits highlighting their structure, function, and between M3 and M4, and an extracellular C-terminus (4–28 expression in a variety of pathological conditions. residues, Fig. 1A) (Corringer et al., 2000). The N-terminus contains the ACh-binding domain (Eisele et al., 1993) with the interface 2. Neuronal nicotinic acetylcholine receptors between adjacent subunits forming a hydrophobic pocket that serves as the agonist-binding site. The M2 transmembrane 2.1. General overview segment of all 5 subunits forms the conducting pore of the channel, with regions in the M1–M2 intracellular loop contributing Signaling through neuronal nAChRs underlies several funda- to cation permeability and agonist-binding affinities (Corringer mental biological processes both during development and in the et al., 2000; McGehee and Role, 1995). When activated by an adult (Albuquerque et al., 2009). In the central nervous system agonist in native or heterologous expression systems, nAChRs (CNS), presynaptic nAChRs modulate release of most classical exhibit a rapid (500 ms to peak current, [ACh] = EC50) inward neurotransmitters including norepinephrine, acetylcholine (ACh), current that desensitizes (Barnard et al., 1982) and is potentiated glutamate and GABA (McGehee et al., 1995). Postsynaptic nAChRs by calcium ions (Vernino et al., 1992). The functional diversity are intimately involved in fast ACh-mediated synaptic transmis- exhibited by the neuronal nAChR family is a consequence, in large sion in addition to activity-dependent gene expression, which is part, of the differential expression of the various subunit genes critical for synaptic plasticity (Albuquerque et al., 2009; Dani and leading to the incorporation of distinct subunits into mature Bertrand, 2007; Hu et al., 2002; Ji et al., 2001). Within the receptors. peripheral nervous system (PNS), nAChRs mediate fast excitatory Much of what is known about the biophysical and pharmaco- transmission in most, if not all, autonomic ganglia and are involved logical properties of nAChRs is based on studies in heterologous in modulating visceral and somatic sensory transmission (Boyd expression systems (McGehee and Role, 1995). These systems et al., 1991; Genzen et al., 2001; Hu and Li, 1997; Steen and Reeh, make use of nAChR mRNA or cRNA injected into Xenopus oocytes as 1993; Sucher et al., 1990; Wang et al., 2002b). More recently, well as nAChR cDNA transfected into mammalian cell lines in order numerous studies have revealed the expression of nAChRs on to express nAChR subunits singly or in combination. The 214 M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226

Fig. 1. nAChR structure. (A) Schematic representation of an individual nAChR subunit. The amino and carboxy terminals of the are labeled N and C, respectively, with the transmembrane segments labeled M1–M4. (B) Examples of homomeric and heteromeric nAChR subtypes. Individual receptor subunits are represented as colored circles. Diamonds located between adjacent receptor subunits represent ligand-binding sites while unfilled circles in the center of each pentamer represent the pore region. (C) Schematic representation of the human CHRNA5/A3/B4 gene cluster. Each gene is drawn to scale with blue boxes representing exons and red boxes representing untranslated regions. Horizontal black lines represent introns while horizontal gray lines represent intragenic regions. The boundaries for each gene are labeled with corresponding Genbank annotations. Colored arrows indicate the direction of transcription. The recently discovered D398N polymorphism (rs16969968) associated with nicotine dependence and lung cancer is indicated by the arrow below the fifth exon of the CHRNA5 gene. propensity for nAChR subunits to form homomeric subtypes was subunit alone (Albuquerque et al., 2009; Karlin, 2002). Thus, for determined by expressing subunits singly. When expressed alone, several years after its initial cloning, the impact of the a5 subunit a7–a10 are able to form functional receptors (Couturier et al., on nAChR function was poorly understood. This mystery was 1990a; Gerzanich et al., 1994). In contrast, other a subunits require solved when a5 was co-expressed with both a4 and b2 subunits the presence of b subunits to form functional receptors (Fig. 1B). (Ramirez-Latorre et al., 1996). ACh-evoked currents from Xenopus Of the clustered receptor subunit genes, co-expression of a3 oocytes expressing the three subunits were distinct from ACh- and b4 nicotinic receptor subunit cRNA results in functional evoked currents in oocytes expressing only a4 and b2 subunits. In receptors with a single channel conductance, g,of24 pS particular, expression of a5 dramatically shifted the ACh (McGehee and Role, 1995; Nelson and Lindstrom, 1999). ACh concentration response curve to the right and significantly and nicotine are less potent agonists of a3b4 nAChRs compared to increased the predominant single channel conductance the prototypic high affinity a4b2 nAChRs (Table 1). In addition, a3 (a4b2 = 24 pS, a4a5b2 = 44 pS at 100 mV). Finally, cysteine can co-assemble with b2 nAChR subunits and form a functional mutagenesis of amino acid residues in the M2 domain of the a5 receptor (Table 1)(Boulter et al., 1987; Wada et al., 1988). Unlike nAChR subunit conferred MTSET sensitivity to a4b2* nAChRs a3, the a5 subunit fails to form functional receptors when co- indicating participation of the a5 subunit M2 domain with the expressed with either b4orb2 subunits (Boulter et al., 1987; pore of assembled receptors (‘‘*’’ denotes containing, Lukas et al., Couturier et al., 1990b). Although the a5 subunit harbors the 1999). Later studies analyzed the impact of a5 expression on a3b2 characteristic viscinal cysteines in the large extracellular domain, a and a3b4 nAChR function. In contrast to its effects on the tyrosine residue (Tyr198) required for ligand-binding has been pharmacology of a4b2nAChRs,a5 expression increased the substituted with an aspartic acid residue, likely explaining the lack sensitivity of a3b2 nAChRs to nicotine and ACh (Table 1)(Wang of functionality of this subunit when expressed with either b et al., 1996). Surprisingly, the ACh/nicotine concentration–response

Table 1 ACh and nicotine potency for various nAChR subtypes.

Subtype EC50 ACh (mM) EC50 Nic (mM) Species Reference

a3b2 26–209 6.8–70 Human Wang et al. (1996); Gerzanich et al. (1998); Nelson et al. (2001) a3a5b2 0.5–121 1.9–83 Human Wang et al. (1996); Gerzanich et al. (1998); Nelson et al. (2001) a3b4 79–180 56–106 Human Wang et al. (1996); Gerzanich et al. (1998); Nelson et al. (2001); Groot-Kormelink et al. (2001) a3a5b4 81–207 42–105 Human Wang et al. (1996); Gerzanich et al. (1998); Nelson et al. (2001); Groot-Kormelink et al. (2001) a4b2b3 0.28 0.26; 48.7 43.1 1.78 0.7 Human Kuryatov et al. (2008) a4a5b2 1.23 0.42 0.36 0.081 Human Kuryatov et al. (2008) a4b2 0.23–1.9 0.12–0.3 Human Kuryatov et al. (2008); Kuryatov et al. (2000) a4b2 0.8 0.8 Chick Ramirez-Latorre et al. (1996) a4a5b2 100 12 Chick Ramirez-Latorre et al. (1996)

Nic = Nicotine; ACh = acetylcholine; EC50 values were taken from studies discussed in the text. M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 215 relationship between a3b4anda3a5b4 nAChRs was found to not are not as sensitive to nicotine-induced inhibition of locomotion dramatically differ (Wang et al., 1996). However, a5 subunit (Salas et al., 2003a, 2004a). b4 KO mice also appear less anxious expression did accelerate the rate of desensitization of these compared to WT mice in two specific anxiety assays suggesting a receptors. In addition, expression of the a5 subunit reduced role for b4* nAChRs in modulating anxiogenic stimuli (Salas et al., channel sensitivity to agonist in a3b4 nAChRs with engineered 2003b). These mice also have a lower core body temperature which point mutations in the M2 domain that renders receptors is less responsive to modulation by acute nicotine infusion (Sack hypersensitive to agonist definitively illustrating that a5 nAChR et al., 2005). subunits can co-assemble with a3b4 nAChRs (Groot-Kormelink et al., 2001). Finally, coassembly of the a5 subunit with a3b2or 3. Regulation of CHRNA5/A3/B4 expression a3b4 nAChRs increases the calcium permeability of the resulting receptors (Gerzanich et al., 1998) indicating that these receptors 3.1. Expression patterns of the CHRNA5/A3/B4 genes could play significant roles in the initiation of ACh-induced signaling cascades under normal and pathological conditions. In Although the initial focus of nAChR expression was in the addition to uncovering a functional role of the a5 nAChR subunit, nervous system, it is now clear that ‘‘neuronal’’ nAChR genes are these studies demonstrated that a portion of functional nAChRs in also expressed in non-neuronal cells where they participate in a native tissue likely consist of heteropentamers containing three or number of fundamental processes (Gahring and Rogers, 2006; more distinct subunits. Sharma and Vijayaraghavan, 2002; Spindel, 2003; Wessler and Kirkpatrick, 2008). This is certainly true for the CHRNA5/A3/B4 2.3. The CHRNA5/A3/B4 gene cluster subunit genes. The CHRNA5/A3/B4 genes are co-expressed in many cell types, thus their clustering may reflect coordinate regulation The CHRNA5/A3/B4 nAChR subunit genes are found in a tight (Table 2). This hypothesis is supported by the fact that the cluster in chromosomal region 15q24–25 (Fig. 1C) (Boulter et al., transcriptional activities of the promoter regions of the three genes 1990). Admixtures of the nAChR subunits encoded by this locus are regulated by many of the same transcription factors (Fig. 2). form the predominant nicotinic receptor subtypes expressed in the However, the CHRNA5/A3/B4 genes are not always co-expressed, PNS (Conroy and Berg, 1995; Covernton et al., 1994; Flores et al., suggesting that independent regulation of each gene also occurs 1996; Rust et al., 1994; Vernallis et al., 1993) as well as at key sites (Table 2 and Fig. 2). in the CNS such as the medial habenula (Gotti et al., 2007; Grady et al., 2009). To determine the function of the clustered nAChR 3.1.1. CHRNA3 subunits, knockout (KO) mice have been generated. Mice that do In the nervous system, the a3 subunit is highly expressed in the not express the a3 subunit usually die within a week of birth due to periphery with a more restricted expression profile in the CNS multi-organ dysfunction (Xu et al., 1999a). a3 KO mice develop (Gotti and Clementi, 2004; Greenbaum and Lerer, 2009). In the enlarged bladders causing bladder infection, dribbling urination, PNS, a3 subunit expression is seen in trigeminal sensory neurons and urinary stones—a phenotype resembling that of a rare human (Flores et al., 1996; Liu et al., 1998), facial motoneurons (Senba condition called megacystis-microcolon-intestinal hypoperistalsis et al., 1990), retina (Feller, 2002; Moretti et al., 2004), dorsal root syndrome (Xu et al., 1999a). Consistently, patients with this ganglia (Zoli et al., 1995) as well as SCG, adrenal medulla, disease do not appear to express a3 mRNA (Richardson et al., spenopalatine and otic ganglia (Rust et al., 1994). Centrally, the a3 2001). a3 KO mice also display extreme pupil dilation and lack of subunit is expressed in the brainstem (Morley, 1997; Wevers et al., pupil contraction in response to light and have retinal wave 1994), cerebellum (Hellstro¨m-Lindahl et al., 1999; Turner and activity with altered spatiotemporal properties delaying the Kellar, 2005), spinal cord (Hellstro¨m-Lindahl et al., 1998; Keiger refinement of retinal ganglion cell dendrites (Bansal et al., 2000; et al., 2003), substantia nigra (Azam et al., 2002), medial habenula Xu et al., 1999a). Bladder contraction in response to nicotine is also (Grady et al., 2009; Zoli et al., 1995), pineal gland (Zoli et al., 1995), lost. In addition, electrophysiological characterization shows that hippocampus (Gahring et al., 2004a; Guan et al., 2002; Hellstro¨m- nicotine-induced whole-cell currents are abolished in the superior Lindahl et al., 1999; Terzano et al., 1998; Winzer-Serhan and Leslie, cervical ganglion (SCG) of a3 KO mice. 1997), cortex (Guan et al., 2002; Hellstro¨m-Lindahl et al., 1999) In contrast to the a3 KO mice, a5 and b4 KO mice are both thalamus (Perry et al., 2002; Terzano et al., 1998), ventral viable and lack any gross abnormalities (Wang et al., 2002a, 2003; tegmental area (Greenbaum and Lerer, 2009; Perry et al., 2002) Xu et al., 1999a). a5 KO mice do exhibit abnormal cardiac and interpeduncular nucleus (Grady et al., 2009; Perry et al., 2002; parasympathetic ganglionic transmission and are less sensitive to Winzer-Serhan and Leslie, 1997). acute nicotine treatment. Loss of a5 selectively affects axonal Outside the nervous system, the a3 subunit is expressed in nAChRs in the SCG while leaving somatodendritic receptors human oral keratinocytes (Arredondo et al., 2001, 2005; Conti- unaffected (Fischer et al., 2005). Similarly, ganglionic transmission Tronconi et al., 1994) where its expression, both mRNA and is impaired in b4 KO mice, attenuating ileal and bladder contractile protein, is increased following exposure to nicotine (Arredondo responses to nicotinic agonists. Nicotine-induced whole-cell et al., 2001, 2005; Zia et al., 2000). a3-containing nAChRs are also currents in the SCG of b4 KO mice are also reduced but still expressed in lymphocytes (Benhammou et al., 2000), the present, suggesting that compensation from another subunit (i.e. gastrointestinal tract (Flora et al., 2000a; Glushakov et al., b2) may be occurring (Xu et al., 1999b). Consistent with this 2004), vascular endothelial cells (Macklin et al., 1998; Wang notion, nicotine-induced currents in the SCG are abolished in et al., 2001), polymorphonuclear cells (Benhammou et al., 2000), double b2–b4 KO mice. Moreover, double b2–b4 KO mice exhibit bronchial epithelium (Maus et al., 1998; Wang et al., 2001) and similar bladder and pupil dysfunction as a3 KO mice. Taken O2A progenitors (Rogers et al., 2001). The a3 subunit is also together, these studies indicate that the clustered nAChR subunits expressed in lung (Improgo et al., 2010; Lam et al., 2007; Sartelet are essential for normal ganglionic function and that compensation et al., 2008) and its expression increases in small cell lung by b2 can occur with the loss of b4. carcinoma (Improgo et al., 2010). In addition to their PNS-specific phenotypes, a3, a5 and b4KO mice also exhibit CNS-centric abnormalities compared to WT mice. 3.1.2. CHRNA5 For example, a3, a5 and b4 KO animals are resistant to nicotine- Similar to the a3 subunit, the a5 subunit is most highly induced seizures compared to their respective WT littermates and expressed in the PNS but is also expressed in several key regions of 216 M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226

Table 2 Expression of the CHRNA5/A3/B4 Genes.

Location CHRNA5 CHRNA3 CHRNB4 Reference

CNS Brainstem + + nd Hellstro¨m-Lindahl et al. (1999); Morley (1997); Wevers et al. (1994) Cerebellum ++ + + Flora et al. (2000a); Hellstro¨m-Lindahl et al. (1999); Winzer-Serhan and Leslie (1997) Cortex + + + Wada et al. (1990); Hellstro¨m-Lindahl et al. (1999); Dineley-Miller and Patrick (1992) Habenula + nd + Grady et al. (2009); Winzer-Serhan and Leslie (1997) Hippocampus +++ + + Hellstro¨m-Lindahl et al. (1998); Wada et al. (1990); Winzer-Serhan and Leslie (1997) Interpeduncular nucleus +++ +++ +++ Grady et al. (2009); Winzer-Serhan and Leslie (1997); Dineley-Miller and Patrick (1992) Medial habenula +++ +++ Boulter et al. (1990); Winzer-Serhan and Leslie (1997) Olfactory bulb nd +++ +++ Dineley-Miller and Patrick (1992) Pineal gland nd +++ +++ Zoli et al. (1995); Dineley-Miller and Patrick (1992) Substantia nigra ++ ++ ++ Azam et al. (2002) Spinal cord + + + Flora et al. (2000a); Hellstro¨m-Lindahl et al. (1998); Zoli et al. (1995) Thalamus + + + Flora et al. (2000a); Hellstro¨m-Lindahl et al. (1998) Ventral tegmental area ++ ++ ++ Azam et al. (2002)

PNS Adrenal medulla nd + + Rust et al. (1994); Di Angelantonio et al. (2003) Dorsal root ganglia + + ++ Flora et al. (2000a); Zoli et al. (1995) Facial motoneurons nd + nd Senba et al. (1990) Otic ganglia nd ++ ++ Rust et al. (1994); Zoli et al. (1995) Retina + + + Moretti et al. (2004) Sphenopalatine ganglia nd ++ + Rust et al. (1994); Zoli et al. (1995) Superior cervical root ganglia + ++ ++ Flora et al. (2000a); Rust et al. (1994); Zoli et al. (1995) Trigeminal ganglia + +++ ++ Liu et al. (1998); Flores et al. (1996)

Non-neuronal Bronchial epithelium + + + Maus et al. (1998); Wang et al. (2001) Gastrointestinal tract ++ + + Flora et al. (2000a) Lung + + + Flora et al. (2000a); Improgo et al. (2010) T lymphocytes nd + + Battaglioli et al. (1998) Oral epithelium + + + Arredondo et al. (2001, 2005) Oral kerotinocytes nd + + Arredondo et al. (2001, 2005) 02A progenitor + + ++ Rogers et al. (2001) Thymus + ++ nd Flora et al. (2000a) Testis + nd nd Flora et al. (2000a) Vascular endothelial cells + + + Macklin et al. (1998); Wang et al. (2001) nd = Not determined; = no expression; + = expression; ++ = intermediate expression; +++ = high levels of expression.

Fig. 2. Transcriptional regulation of the CHRNA5/A3/B4 gene cluster. (A) Protein–DNA interactions regulate expression of the clustered nAChR subunit genes. Coding regions of the subunits are represented as light green boxes with arrows indicating the direction of transcription. Two transcriptional regulatory elements, the intronic repressor in the fifth intron of a5 (A3I5) and the 30 enhancer in the b4 gene, are shown (green and purple boxes, respectively). Horizontal black lines depict the noncoding regions of DNA. Transcription factors that regulate expression of these genes are depicted as colored circles and are touching each other if they directly interact. Circles labeled with a ‘‘?’’ indicate transcription factors whose identities have yet to be identified. In the case of Sp1, multiple binding sites have been identified in each of the nAChR genes, however, for clarity, the sites are represented as a single green circle at each promoter region. (B) Positive and negative regulation of the clustered nAChR subunit genes. Coding regions of the clustered subunits are represented as light green boxes with arrows that indicate the direction of transcription. Four transcriptional regulatory elements are depicted in this figure: A3I5 (green box), the b430 enhancer (purple), the SacI–HindIII fragment of the b4 promoter region (orange box), and the distal CNR4 regulatory region (yellow box). The boundaries of the latter two regions are labeled relative to the transcriptional start site of the b4 gene. Red arrows denote positive regulatory effects whereas blue arrows indicate negative transcriptional regulation. M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 217 the CNS (Gotti and Clementi, 2004; Greenbaum and Lerer, 2009). 3.2. Transcriptional regulation of the CHRNA5/A3/B4 genes Centrally, a5 is expressed primarily in the cerebellum and thalamus (Flora et al., 2000a) but is also detected in the cortex, The co-expression of the CHRNA5/A3/B4 genes coupled with hippocampus, brainstem, spinal cord, habenula, interpeduncular their genomic clustering suggests they may share common nucleus and other midbrain nuclei (Azam et al., 2002; Gahring regulatory mechanisms in addition to specific regulation of each et al., 2004a; Grady et al., 2009; Hellstro¨m-Lindahl et al., 1998; gene. Further support for this idea comes from several observa- Keiger et al., 2003; Wada et al., 1990; Zoli et al., 2002). In the PNS, tions. First, nucleotide sequencing of the individual gene promo- a5 is expressed in most autonomic ganglia (Flora et al., 2000a; Liu ters revealed that they each lack classical CAAT and TATA boxes et al., 1998) and the retina (Moretti et al., 2004). (Boulter et al., 1990). Instead, the promoters are GC-rich and In addition to the nervous system, a5 subunit expression has contain several binding sites for the transcription factors, Sp1 and been detected in the gastrointestinal tract, where its expression is Sp3 (Fig. 2, Table 3). Both Sp factors positively regulate substantially higher than that of the a3 subunit, thymus and testis transcription of each of the clustered subunit genes through (Flora et al., 2000a; Glushakov et al., 2004). Furthermore, the a5 multiple binding sites in each individual promoter (Bigger et al., subunit is expressed in many of the same cell types as the a3 and 1996, 1997; Boyd, 1996; Campos-Caro et al., 2001; Campos-Caro b4 subunits including oral epithelium (Arredondo et al., 2001, et al., 1999; Flora et al., 2000b; Melnikova and Gardner, 2001; 2005), vascular endothelial cells (Macklin et al., 1998; Wang et al., Melnikova et al., 2000a; Terzano et al., 2000; Valor et al., 2002; 2001), bronchial epithelium (Maus et al., 1998; Wang et al., 2001), Yang et al., 1995). Chromatin immunoprecipitation (ChIP) experi- O2A progenitors (Rogers et al., 2001) and a variety of immune cells ments demonstrated Sp1 binding activity in the context of native (Wessler and Kirkpatrick, 2008). chromatin for all three promoters (Benfante et al., 2007; Scofield et al., 2008). It is likely that Sp1 is involved in tethering the basal 3.1.3. CHRNB4 transcription machinery to the TATA-less nAChR subunit gene As with the other two genes in the nAChR cluster, the b4 promoters (Pugh and Tjian, 1991). Second, in addition to the Sp subunit gene is widely expressed in the PNS with more limited factors, the CHRNA5/A3/B4 promoter regions can directly interact expression centrally (Gotti and Clementi, 2004). b4 expression is with and be trans-activated by the more spatially restricted relatively high in trigeminal sensory neurons (Flores et al., 1996; regulatory factors Sox10 and SCIP/Tst-1/Oct-6 (Fig. 2)(Fyodorov Liu et al., 1998) as well as the superior cervical, dorsal root, and Deneris, 1996; Liu et al., 1999; Yang et al., 1994). Third, the spenopalatine and otic ganglia and sympathetic neurons (Man- mRNA levels of the CHRNA5/A3/B4 genes are coordinately up- delzys et al., 1994; Rust et al., 1994; Zoli et al., 1995). The b4 regulated during neural development (Corriveau and Berg, 1993; subunit is also expressed in the adrenal medulla (Di Angelantonio Levey et al., 1995; Levey and Jacob, 1996) and coordinately down- et al., 2003) with lower expression in the retina (Moretti et al., regulated following denervation (Zhou et al., 1998). Perhaps the 2004). In the CNS, b4 expression is particularly high in the most compelling evidence for a coordinated regulatory scheme olfactory bulb, pineal gland, medial habenula and interpeduncular comes from the Deneris lab, which showed that two transcrip- nucleus (Dineley-Miller and Patrick, 1992; Grady et al., 2009; tional regulatory elements, b430 and conserved noncoding region 4 Winzer-Serhan and Leslie, 1997) with lower expression in other (CNR4), play key roles in directing expression of the clustered thalamic nuclei, the cortex, hippocampus, spinal cord, cerebellum nAChR genes in a tissue-specific manner with b430 being and midbrain (Azam et al., 2002; Gahring et al., 2004a; Hellstro¨m- important for expression in the adrenal gland and CNR4 being Lindahl et al., 1998; Keiger et al., 2003; Perry et al., 2002; Quik et al., critical for expression in the pineal gland and superior cervical 2000; Turner and Kellar, 2005). ganglion (Fig. 2)(Xu et al., 2006). CNR4 is likely to play an Outside the nervous system, there is again significant overlap of important role in directing nAChR gene expression in the brain as b4 expression with a3 and a5 expression. b4 is expressed in well (Xu et al., 2006). In addition to these shared regulatory multiple cell types of the intestine (Flora et al., 2000a; Glushakov features, the CHRNA5/A3/B4 genes are subject to gene-specific et al., 2004), vascular endothelial cells (Macklin et al., 1998), oral regulation. keratinocytes (Arredondo et al., 2005; Arredondo et al., 2001), polymorphonuclear cells (Benhammou et al., 2000), bronchial 3.2.1. CHRNA3 epithelium (Maus et al., 1998; Wang et al., 2001) and O2A In vitro experiments have shown that the paired-like home- progenitors (Rogers et al., 2001). Finally, b4 is co-expressed with odomain transcription factor, PHOX2A, regulates transcription a3 and a5 in lung (Improgo et al., 2010; Lam et al., 2007; Sartelet from the a3 promoter (Benfante et al., 2007). PHOX2A does not et al., 2008) and is also up-regulated in lung cancer (see below) appear to bind directly to DNA, however, as the DNA-binding (Improgo et al., 2010). domain does not need to be completely intact for PHOX2A to

Table 3 Transcription factors that regulate the CHRNA5/A3/B4 genes.

Transcription Factor CHRNA5 CHRNA3 CHRNB4 Reference

ASCL1 nd Yes Yes Improgo et al. (2010) Brn-3a nd Yes No Milton et al. (1996) c-Jun nd nd Yes Melnikova and Gardner (2001) hnRNP K nd nd Yes Du et al. (1998) NF-Y nd nd Yes Valor et al. (2002) Pet-1 nd Yes nd Fyodorov et al. (1998) PHOX2A nd Yes nd Benfante et al. (2007) Pura nd nd Yes Du et al. (1997) SCIP nd Yes Yes Fyodorov and Deneris (1996); Yang et al. (1994) Sox10 Yes Yes Yes Liu et al. (1999) Sp1 Yes Yes Yes Campos-Caro et al. (1999); Yang et al. (1995); Bigger et al. (1996) Sp3 Yes Yes Yes Bigger et al. (1997) nd = Not determined; no = no regulation; yes = regulation by the indicated transcription factor. 218 M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 regulate transcription from the a3 promoter (Benfante et al., the expression of a3 and b4 and modestly of a5 in lung cancer cells 2007). Co-immunoprecipitation experiments demonstrate a phys- (Improgo et al., 2010). ical interaction between Sp1 and PHOX2A, suggesting that PHOX2A is tethered to the a3 promoter through its interaction 3.2.2. CHRNA5 with Sp1, similar to the interactions of Sp1 with homeodomain The a5 promoter region has been described in several genomic transcription factors observed in other systems (Shimakura et al., contexts including those in rodents and humans. Transcription of 2006). a5 occurs in the opposite direction as a3 and b4(Fig. 1), As briefly described above, the POU domain factor SCIP/Tst-1/ suggesting that in addition to transcription factors that regulate Oct-6 has been shown to positively regulate transcription from the the entire cluster, distinct mechanisms may govern a5 expression. a3 promoter in a cell-type-specific manner (Yang et al., 1994). However, apart from the regulatory factors described above that Similar to PHOX2A, the POU domain factor SCIP/Tst-1/Oct-6 does control expression of all three clustered genes, little is known not require DNA-binding for trans-activation of the a3 promoter about these mechanisms. SCIP/Tst-1/Oct-6 does not appear to (Yang et al., 1994). Deletional analysis of the SCIP/Tst-1/Oct-6 regulate a5 though it regulates a3 and b4. Similarly, in lung cells, transcription factor demonstrated that only the POU domain is ASCL1 appears to regulate a3 and b4 but not a5(Improgo et al., needed for trans-activation. Interestingly, this trans-activation does 2010). No other transcription factors regulating a5 expression not depend on the presence of an Sp1 motif in the promoter region have been reported, underscoring the need for more research effort and is likely occurring through protein–protein interactions with the in this area. basal transcription machinery (Fyodorov and Deneris, 1996). The transcription factor Brn-3a also trans-activates the a3promoter, 3.2.3. CHRNB4 while the other members of the Brn-3 family, Brn-3b and 3c, In addition to the Sp factors, Sox10 and SCIP/Tst-1/Oct-6, the b4 modestly repress a3 promoter activity (Milton et al., 1996). The promoter is positively regulated by c-Jun (Melnikova and Gardner, positive regulation by Brn-3a is thought to be a result of protein– 2001). Trans-activation by all of these factors is abolished when the protein interaction as the a3 promoter lacks an obvious octamer or Sp-binding site on the b4 promoter (referred to as a CA box) is octamer-related binding site for Brn-3 factors (Milton et al., 1996). mutated. Conversely, synergistic activation of the b4 promoter is A transcriptional enhancer has been discovered upstream of the observed when Sp1 is supplied in concert with Sox10, Sp3 or c-Jun a3 promoter in a region that overlaps with a 30-untranslated exon (Melnikova and Gardner, 2001; Melnikova et al., 2000a). Co- of the b4 gene (McDonough and Deneris, 1997). This transcrip- immunoprecipitation experiments demonstrated that all of these tional enhancer consists of two identical 37- repeats factors physically interact (Melnikova et al., 2000b) and ChIP separated by a 6-base pair spacer. The b430 enhancer acts as a cell- experiments confirmed that these interactions occur in the context type-specific enhancer and is capable of enhancing transcription of native chromatin (Scofield et al., 2008). These findings suggest from the a3 promoter in neuronal-like cells, as well as in neurons the existence of a positively acting multi-subunit transcriptional in SCG cultures (McDonough et al., 2000). The enhancer contains regulatory complex that assembles on the b4 promoter. This result several E-twenty six (ETS) factor-binding sites, the mutation of is consistent with the hypothesis that Sp1 is critical for which dramatically decreases, but does not completely abolish, a3 transcription from the b4 promoter and likely nucleates the promoter activity. The ETS-domain binding factor, Pet-1, has been regulatory complex that drives expression of the b4 gene. shown to activate reporter gene transcription in a manner that is Two additional transcription factors have been shown to both cell type- and b430 enhancer-dependent (Fyodorov et al., interact with the b4 promoter, Pura and heterogeneous nuclear 1998). Taken together, these experiments suggest that Pet-1 ribonucleoprotein K (hnRNP K) (Du et al., 1997, 1998). These interacts directly with the a3 promoter to activate transcription, proteins interact with another motif, the CT box, located directly though it likely requires additional cell-type-specific co-factors. In upstream of the CA box. hnRNP K is capable of repressing Sp factor- vivo experiments using transgenic mice showed that a larger DNA mediated trans-activation of the b4 promoter (Du et al., 1998) and fragment between the a3 and b4 genes, containing both the b430 also physically interacts with Sox10 (Melnikova et al., 2000b). enhancer and the a3 promoter, is capable of directing expression Similar to hnRNP K, Pura physically interacts with Sox10 of a reporter gene to several areas of endogenous a3 expression in (Melnikova et al., 2000b). Moreover, Pura and hnRNP K themselves the brain (Wada et al., 1989; Yang et al., 1997). Surprisingly physically interact (Melnikova et al., 2000b). These proteins may however, this DNA fragment did not direct reporter gene participate in the multi-subunit complex described above to expression anywhere in the peripheral nervous system, in which modulate expression of the b4 gene in the appropriate cellular the a3 gene is highly expressed (see above), suggesting that context. In vitro binding experiments demonstrated that each elements in this fragment may be acting as repressors or that other factor binds preferentially to the opposing single strand elements sequences are necessary for peripheral expression. of the CT box, suggesting that some local DNA helix unwinding The presence of an intronic repressor element in the fifth intron may occur (Krecic and Swanson, 1999). Interestingly, Pura and of a3 has in fact been reported (Fuentes Medel and Gardner, 2007). hnRNP K have been shown to function together to negatively The sequence of this a3 intron 5 repressor (a3I5) is highly impact transcription of genes in other systems and the same may conserved and is capable of bidirectional repressor activity in vitro. be occurring at the b4 promoter (Da Silva et al., 2002). In vivo Notably, cell-type-specific repression of promoter activity was experiments have also shown that a 2.3-kb fragment of the b4 observed to be more potent in non-neuronal cell lines than in promoter, containing the CA and CT boxes, is capable of directing neuronal cell lines (Fuentes Medel and Gardner, 2007). These data reporter gene expression to brain regions that endogenously suggest that this segment of DNA and the factors with which it express b4, further supporting the importance of these elements in interacts function to restrict expression of a3 to neuronal cell regulating b4 gene expression (Bruschweiler-Li et al., 2010). types. The protein–DNA interactions that mediate this effect have yet to be elucidated. 4. Role in pathological states While regulation of the a3 subunit gene in neuronal cells has been extensively studied, the mechanisms regulating a3 expres- 4.1. Nicotine addiction sion in non-neuronal cells remain largely obscure. Recently, however, our group was the first to report that a transcription Nicotine addiction is responsible for approximately 5 million factor, the achaete–scute complex homolog-1 (ASCL1), regulates deaths per year worldwide and is the most preventable cause of M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 219 death in the USA (CDC, 2008). Nicotine addiction involves a series of nicotine (Sherva et al., 2008; Stevens et al., 2008). In addition, the of events starting with the initial use of cigarettes, the transition presence of this SNP may also influence the biophysical properties from experimental smoking to regular smoking, and finally of a5* nAChRs. When expressed in HEK293T cells, a4b2a5D398 dependence on nicotine (Bierut, 2009b). Nicotine dependence is nAChRs exhibited a greater maximal response to nicotine characterized by heavier smoking, early morning smoking, compared to a4b2a5N398 nAChRs as measured by calcium tolerance and withdrawal. Withdrawal symptoms account for imaging (Bierut et al., 2008). The association of this particular SNP the high incidence of relapse in people attempting to quit smoking has been identified in several studies though the majority of them (Kenny and Markou, 2001). have involved populations of European descent. In populations of At the molecular level, nicotine addiction is initiated by the African and Asian descent, the risk allele of rs16969968 is rare but binding of nicotine to nAChRs. This interaction results in increased is still associated with nicotine dependence (Saccone et al., 2009b; dopamine (DA) release in the DAergic mesolimbic and mesocor- Wu et al., 2009). tical circuits (Dani and De Biasi, 2001; Dani and Heinemann, 1996), How the clustered nAChRs function in the development of a phenomenon widely associated with reward or reinforcement addiction is still unknown though recent studies in nAChR KO mice and drugs of abuse. DAergic projection neurons in the mesocorti- have revealed a potential role for these subunits in nicotine colimbic pathway originate in the ventral tegmentum area (VTA) withdrawal. Withdrawal symptoms can be divided into two and project to the nucleus accumbens in the ventral striatum and classes: somatic or physical symptoms, which likely involve both the prefrontal cortex (Laviolette and van der Kooy, 2004). Several the PNS and CNS; and affective symptoms, which are centrally nAChR subtypes are robustly expressed in DAergic neurons both at mediated and are associated with hypoactivity of DAergic neurons the level of the soma, as well as at presynaptic terminals. Although, in the reward circuit (Fung et al., 1996). As in humans, rodents of the clustered nicotinic receptor subunit genes, a5 has been chronically exposed to nicotine exhibit both somatic and affective clearly identified as a component of nAChRs at DAergic terminals withdrawal behaviors upon cessation. Somatic withdrawal symp- (Grady et al., 2007). Much of what is known regarding nAChR toms in rodents are characterized by increased licking, grooming, subtypes that are involved in nicotine addiction stems from studies shaking and scratching (Damaj et al., 2003; Kenny and Markou, on genetically engineered KO or knock-in mice (Champtiaux and 2001; Malin et al., 1994). Rodent affective symptoms include Changeux, 2004). Mice that lack expression of b2* nAChRs fail to increased anxiety, hypolocomotion, and conditioned place aversion self-administer nicotine and exhibit impaired tolerance to nicotine to nicotinic receptor antagonists (Damaj et al., 2003; Jackson et al., (Maskos et al., 2005; McCallum et al., 2006; Picciotto et al., 1998). 2008a; Suzuki et al., 1996). The initiation and expression of In addition, the predominant partner of the b2 nAChR subunit, a4 withdrawal is dependent on neuronal nAChRs based on the fact has also been implicated in nicotine dependence (Marubio et al., that (1) symptoms can be alleviated by nicotine treatment and (2) 2003; Tapper et al., 2004, 2007). Selective activation of a4* nAChRs symptoms can be precipitated by administration of nicotinic by low doses of nicotine in mice expressing a4* nAChRs receptor antagonists during chronic nicotine exposure. Mecamyl- hypersensitive to agonist is sufficient for nicotine reward/ amine, a noncompetitive nicotinic antagonist, precipitates somatic reinforcement, sensitization, and tolerance (Tapper et al., 2004). withdrawal symptoms as well as affective symptoms including More recently, it was found that mice that do not express a6* hypolocomotion, place aversion and anxiety, when injected i.p. in nAChRs fail to self-administer nicotine (Pons et al., 2008). nicotine-dependent mice or rats (Damaj et al., 2003; Malin et al., Expression of the a6 subunit has also been observed in the 1994; Suzuki et al., 1996, 1999). The more selective high affinity striatum and about half of a6* nAChRs co-assemble with a4to nAChR competitive antagonist, dihydro-b-erythroidine (DHbE) form receptors with the highest affinity for ACh in the striatum (Harvey and Luetje, 1996; Harvey et al., 1996), can also precipitate (Champtiaux et al., 2002; Salminen et al., 2007). Because of their both somatic and affective symptoms suggesting a role for high high expression in the reward pathway and high affinity for affinity nAChRs in withdrawal (Malin et al., 1998). Methyllycaco- agonist, a4b2* nAChRs have been studied extensively in the nitine (MLA), a relatively selective a7 antagonist (Alkondon et al., context of nicotine addiction. 1992; Palma et al., 1996; Ward et al., 1990)(butsee(Klink et al., In recent years, linkage analyses, candidate-gene analyses, and 2001)), precipitates mild somatic, but few affective withdrawal large-scale genome-wide association studies (GWAS) have been symptoms (Markou and Paterson, 2001). Finally, somatic with- used to screen hundreds of thousands of SNPs across thousands of drawal symptoms can be precipitated by hexamethonium, a individuals in search of genetic variants associated with nicotine nicotinic antagonist that does not cross the blood brain barrier dependence (Baker et al., 2009; Berrettini et al., 2008; Bierut, suggesting that peripheral nAChRs also play a role in these 2009a; Bierut et al., 2008; Caporaso et al., 2009; Chen et al., 2009a; symptoms (Hildebrand et al., 1997) although similar symptoms can Freathy et al., 2009; Greenbaum and Lerer, 2009; Grucza et al., be elicited by injection of antagonist directly into the brain (Salas 2008; Le Marchand et al., 2008; Saccone et al., 2007, 2009a,b; et al., 2009). Conversely, affective symptoms are not blocked by i.p. Schlaepfer et al., 2008; Schwartz et al., 2009; Spitz et al., 2008; injection of hexamethonium indicating that they are mediated by Stevens et al., 2008; Thorgeirsson et al., 2008; Vink et al., 2009; central nAChRs (Hildebrand et al., 1997; Watkins et al., 2000). Wang et al., 2009a; Weiss et al., 2008). This has led to the Chronic nicotine-treated a5 and b4 KO mice display signifi- identification of multiple SNPs in 15q24–25, a region cantly milder mecamylamine-precipitated somatic withdrawal that contains the CHRNA5/A3/B4 gene cluster as well as the iron- symptoms compared to WT mice (Salas et al., 2004b, 2009). In the responsive element binding protein, a putative protein of unknown CNS, a5 and b4* nAChRs are robustly expressed in the habenulo- function, LOC123688, and the a4 proteasome subunit protein. Of interpeduncular pathway and direct infusion of mecamylamine particular interest is the non-synonymous SNP, rs16969968, found into either the medial habenula or the interpeduncular nucleus can in exon5 of the a5 gene. This polymorphism changes an aspartic precipitate withdrawal in mice dependent on nicotine suggesting acid residue into asparagine at position 398 (D398N) in the second that the nAChRs in this pathway are critical for the expression of intracellular loop of a5. Individuals with one copy of the risk nicotine withdrawal (Salas et al., 2009). However, chronic nicotine- variant have a 1.3-fold increased risk for nicotine dependence treated a5 KO mice still become anxious during withdrawal while individuals with two copies have almost a 2-fold increase in suggesting that a5* nAChRs may not be involved in affective risk. This SNP appears to influence different aspects of nicotine symptoms (Jackson et al., 2008b). dependence, as it has been associated with increased risk for heavy To date, the role of either a5* or b4* nAChRs in nicotine reward smoking as well as increased sensitivity to the pleasurable effects and reinforcement is unclear. Nicotine conditions a place prefer- 220 M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 ence in a5 KO mice indicating that expression of a5* nAChRs may protein kinase, leading to the activation of the oncogenic not be necessary for the rewarding properties of the drug (Jackson transcription factor c-myc, a process that can be blocked by the et al., 2010). However, these animals not only exhibit a preference a7-selective antagonist, a-bungarotoxin (Jull et al., 2001). In for nicotine at similar doses as WT mice, but also at high doses that addition, nicotine and NNK play a role in apoptotic inhibition. normally fail to condition a place preference. Additional analysis of Nicotine activates the anti-apoptotic protein BCL-2 and inactivates the reinforcing properties of nicotine in KO mice that do not the proapoptotic proteins Bad and Bax (Jin et al., 2004; Mai et al., express a5orb4 nAChRs and/or in knock-in mice that express the 2003; Xin and Deng, 2005). Nicotine and NNK also activate the Akt/ a5 polymorphism identified in the GWAS should yield valuable protein kinase B pathway, inhibiting apoptosis and causing insight into the molecular mechanisms underlying nicotine tumorigenesis (West et al., 2003). Activation of Akt by nicotine addiction. appears to depend on nAChRs containing a3ora4 whereas NNK activation depends on a7 nAChRs. Finally, nicotine acts as a pro- 4.2. Lung cancer angiogenic agent, promoting endothelial cell migration, prolifera- tion, tube formation and survival, an effect antagonized by a- Lung cancer is the leading cause of cancer-related deaths for bungarotoxin (Cooke and Ghebremariam, 2008; Heeschen et al., both men and women worldwide (ACS, 2009). In terms of 2001). Along with its metabolite, cotinine, nicotine also up- incidence, lung cancer is second only to prostate cancer in men regulates the expression of the vascular endothelial growth factor and breast cancer in women. Lung cancer can be divided into two in endothelial cells (Conklin et al., 2002). main histological types: small cell lung carcinoma (SCLC) and non- The aforementioned candidate-gene analyses and GWAS have small cell lung carcinoma (NSCLC). The latter can be further also implicated the CHRNA5/A3/B4 gene cluster in lung cancer. The divided into adenocarcinoma, squamous cell, bronchioalveolar and same SNP associated with nicotine dependence was found to large cell lung carcinoma. increase the risk for lung cancer (Amos et al., 2008; Hung et al., Tobacco intake is the main risk factor associated with lung 2008; Thorgeirsson et al., 2008). In particular, Hung and colleagues cancer. The scientific link between tobacco and lung cancer has analyzed approximately 317,000 SNPs in thousands of patients and been firmly established since the 1950s (Proctor, 2001). This controls and found that the non-synonymous SNP, rs16969968, is linkage is not surprising given (1) the addictive effects of nicotine robustly associated with lung cancer. This association was not that promote continued use of tobacco despite health conse- affected by smoking status and was observed even in never- quences and (2) the presence of at least 55 carcinogens in tobacco, smoker groups, suggesting that the association is not due to the including nicotine metabolites such as 4-(methylnitrosamino)-1- influence of nicotine dependence. Furthermore, no increased risk (3-pyridyl)-butanone (NNK) and N-nitrosonornicotine (NNN) for other smoking-related cancers such as head and neck cancers (Hecht, 1999; Shields, 2002). NNK and NNN form DNA adducts was observed, indicating that the association with lung cancer is causing mutations that initiate cancer (Hecht and Hoffmann, direct (Hung et al., 2008). Two other SNPs, in exon 5 and the 30- 1988). Interestingly, these nitrosamines also serve as ligands for untranslated region of the a3 gene, were also found in these nAChRs (Schuller and Orloff, 1998) and along with nicotine and studies to be associated with lung cancer (Amos et al., 2008; Hung ACh, have been shown to trigger a variety of cancer-related et al., 2008; Thorgeirsson et al., 2008). processes as described below. In 1989, Schuller’s group first showed that nicotine and its 4.3. Other diseases metabolites stimulates the growth of lung cancer cells (Schuller, 1989). Shortly thereafter, Minna’s group reported that nicotine Aside from nicotine addiction and lung cancer, the CHRNA5/ causes apoptotic inhibition (Maneckjee and Minna, 1990). These A3/B4 locus has also been associated with alcoholism. SNPs in this two groups and many others thereafter have reported the locus are associated with alcohol dependence and the level of expression of nAChRs in both normal and malignant lung cells, response to alcohol (Chen et al., 2009b; Wang et al., 2009b). supporting the notion that nicotine stimulates cancer-related Furthermore, the variants appear to influence age at initiation of processes in a receptor-mediated fashion (Maneckjee and Minna, both tobacco and alcohol use (Schlaepfer et al., 2008). Since 1990; Maus et al., 1998; Sartelet et al., 2008; Schuller, 1989; Song alcohol and nicotine are usually co-abused, these associations et al., 2003; Wang et al., 2001). Moreover, studies have shown that may provide a genetic mechanism for this co-morbidity (Green- specific nAChR subunits are over-expressed in lung cancer tissue baum and Lerer, 2009). The risk allele of the non-synonymous (Lam et al., 2007). In particular, the CHRNA5/A3/B4 gene cluster is SNP, rs16969968, also appears to be protective for cocaine over-expressed in SCLC and this over-expression appears to be dependence, suggesting a more general involvement of the regulated by ASCL1 (Improgo et al., 2010). ASCL1 is a basic helix- CHRNA5/A3/B4 gene cluster in the actions of drugs of abuse loop-helix transcription factor important in the initiation and (Grucza et al., 2008). development of SCLC (Ball et al., 1993; Jiang et al., 2009; Linnoila Other smoking-related diseases are influenced by variants in et al., 2000; Osada et al., 2005). Regulation of the CHRNA5/A3/B5 the CHRNA5/A3/B4 locus. Two SNPs in this region are associated locus by ASCL1 suggests a mechanism by which over-expression of with chronic obstructive pulmonary disease, a serious lung disease ASCL1 in SCLC leads to a corresponding increase in expression of characterized by chronic inflammation and progressive destruc- the clustered nAChR subunits, thereby potentiating the prolifer- tion of lung tissues (Pillai et al., 2009). Interestingly, nicotine has ative and pro-survival effects of nicotine and other nAChR ligands been shown to promote the development of SCLC-like tumors in a (Improgo et al., 2010). rodent model of chronic obstructive pulmonary disease (Schuller Different groups have shown that various nAChR ligands et al., 1995). Another study showed that a variant influencing the activate distinct cancer-signaling pathways via the a3b2, a3b4, risk for nicotine dependence and lung cancer also increases the risk a4b2 and a7 nAChR subtypes (Schuller, 2009). ACh stimulates cell for peripheral arterial disease, a condition characterized by proliferation by acting as an autocrine growth factor in SCLC, a obstruction of arteries outside the heart and brain (Thorgeirsson phenomenon that can be blocked by the non-specific nAChR et al., 2008). The association of variants in the CHRNA5/A3/B4 gene antagonist, mecamylamine (Song et al., 2003). Nicotine also up- cluster on other smoking-related diseases may be an effect of regulates the expression of growth factors and their cognate nicotine dependence, suggesting a gene-environment interaction, receptors (Conti-Fine et al., 2000). NNK causes cell proliferation by or may represent a direct effect, indicating pleiotropy of this locus activating the serine/threonine kinase RAF1 and mitogen-activated (Bierut, 2009a; Thorgeirsson et al., 2008). M.R.D. Improgo et al. / Progress in Neurobiology 92 (2010) 212–226 221

5. Conclusions association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat. Genet. 40, 616–622. Arneric, S.P., Holladay, M., Williams, M., 2007. Neuronal nicotinc receptors: a Tobacco use continues to be a major global health threat, perspective on two decades of drug discovery research. Biochem. Pharmacol. underscoring the need to understand the mechanisms leading to 74, 1092–1101. Arredondo, J., Chernyavsky, A.I., Marubio, L.M., Beaudet, A.L., Jolkovsky, D.L., Pin- nicotine dependence and smoking-related diseases such as lung kerton, K.E., Grando, S.A., 2005. Receptor-mediated tobacco toxicity: regulation cancer. Nicotine activation of nAChRs in the CNS initiates nicotine of gene expression through a3b2 nicotinic receptor in oral epithelial cells. Am. dependence. However, it is clear that nAChRs are also expressed J. Pathol. 166, 597–613. outside the nervous system and are likely involved in numerous Arredondo, J., Nguyen, V.T., Chernyavsky, A.I., Jolkovsky, D.L., Pinkerton, K.E., Grando, S.A., 2001. A receptor-mediated mechanism of nicotine toxicity in oral smoking-related pathologies. Although most studies to date have keratinocytes. Lab. Invest. 81, 1653–1668. implicated the high affinity a4b2* nAChRs in nicotine dependence, Azam, L., Winzer-Serhan, U.H., Chen, Y., Leslie, F.M., 2002. Expression of neuronal less is known regarding the role of a3a5b4* nAChRs in this disease nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. J. Comp. Neurol. 444, 260–274. (Picciotto et al., 1998; Tapper et al., 2004). However, the recent Baker, T.B., Weiss, R.B., Bolt, D., von Niederhausern, A., Fiore, M.C., Dunn, D.M., Piper, flurry of GWAS implicating the CHRNA5/A3/B4 gene cluster in M.E., Matsunami, N., Smith, S.S., Coon, H., McMahon, W.M., Scholand, M.B., nicotine dependence lends greater credence to the involvement of Singh, N., Hoidal, J.R., Kim, S.Y., Leppert, M.F., Cannon, D.S., 2009. Human neuronal acetylcholine receptor A5–A3–B4 haplotypes are associated with these hitherto understudied nAChR subunits in nicotine addiction multiple nicotine dependence phenotypes. Nicotine Tob. Res. 11, 785–796. (Berrettini et al., 2008; Bierut et al., 2008; Portugal and Gould, Ball, D.W., Azzoli, C.G., Baylin, S.B., Chi, D., Dou, S., Donis-Keller, H., Cumaraswamy, 2008; Saccone et al., 2007, 2009a; Schlaepfer et al., 2008). That A., Borges, M., Nelkin, B.D., 1993. Identification of a human achaete–scute homolog highly expressed in neuroendocrine tumors. Proc. Natl. Acad. 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Sci. 215, 241–246. Battaglioli, E., Gotti, C., Terzano, S., Flora, A., Clementi, F., Fornasari, D., 1998. the polymorphisms identified in the GWAS. Expression and transcriptional regulation of the human alpha3 neuronal nico- Even more provoking are similar studies implicating this gene tinic receptor subunit in T lymphocyte cell lines. J. Neurochem. 71, 1261–1270. cluster in lung cancer (Amos et al., 2008; Hung et al., 2008; Benfante, R., Flora, A., Di Lascio, S., Cargnin, F., Longhi, R., Colombo, S., Clementi, F., Fornasari, D., 2007. Transcription factor PHOX2A regulates the human a3 Thorgeirsson et al., 2008). It is still somewhat controversial, nicotinic receptor subunit gene promoter. J. Biol. Chem. 282, 13290–13302. however, as to whether the association with lung cancer is direct Benhammou, K., Lee, M., Strook, M., Sullivan, B., Logel, J., Raschen, K., Gotti, C., or merely a by-product of the strength of the addiction or smoking Leonard, S., 2000. 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