We have studied global gene expression in bronchial, nasal, and buccal epithelial cells in never and current smokers. Our findings suggest that similar functional categories of genes are expressed in nasal and bronchial epithelial cells of healthy never smokers. We have shown that there are similarities between the effect of smoking on bronchial epithelial gene expression and the gene expression response to smoking in buccal and nasal epithelium. This implies the potential to study disease-relevant responses to tobacco smoke in any of these tissues. This represents a significant advantage as buccal and nasal mucosa can be readily collected from large study cohorts as a result of their ability to be collected non-invasively. Given the burden of smoking-related disease, there is a need for non-invasive biomarkers of the individual-level variability in host responses to cigarette smoke.
The similar pattern of gene expression in bronchial and nasal epithelial cells of never smokers suggests a shared architecture and function. The nasal passage and bronchus are both lined with ciliated pseudostratified columnar epithelial cells, and some of the functions of genes that are highly expressed in both nasal and bronchial epithelial cells are likely due to this common cellular architecture. For example, cilia-related genes such as DNAH7, DNAH9, and DNAI2 were highly expressed in both bronchial and nasal airway epithelium. Consistent with this finding, previous studies have shown that normal ciliated airway epithelial cells express these genes [20–22]. Other dynein light chain genes such as DYNLRB1 which have been characterized in other non-epithelial tissues  were found to be specifically expressed in bronchial epithelial cells, while dynein light chain genes such as DYNLL1 shared relatively high expression specifically in nasal epithelium as well as non-epithelial tissues. Many genes involved in host defense are also expressed at high levels in extra- and intrathoracic airway epithelial cells. Glutathione expression has been previously well characterized in bronchial epithelium as well as in the lung . Our data show high expression of glutathiones such as GSTM1 in bronchial, nasal, and buccal epithelium relative to other non-airway epithelial cells. We also observed high expression of mucins such as MUC1, MUC4, and MUC5AC in bronchial and nasal epithelium and somewhat lower expression of MUC1 in buccal mucosa and lung tissue. Expression of these genes has been well documented in respiratory tract epithelium [25–29]. We found the genes belonging to the cytochrome P450 family and several aldehyde dehydrogenase genes  are highly expressed the bronchial and nasal epithelium. Cytochrome P450 genes have previously been shown to be expressed highly in both bronchial epithelial cells [31–33] and nasal mucosa . Our global analysis of gene-expression of the airway epithelium of healthy never smokers recapitulates gene expression patterns previously observed within these tissue types, thereby lending support to the similarities and differences between tissues that are suggested by our analysis of gene expression in the normal intrathoracic airway transcriptome.
Smoking altered the expression of a set of genes in bronchial epithelial cells which were also commonly altered in nasal and buccal epithelial cells. Gene set enrichment analysis of 361 smoking-induced bronchial genes yielded a subset of genes that were among the most up-regulated by smoking in the mouth (74 leading edge genes), as well as a subset of genes that are among the most up-regulated by smoking in the nose (120 leading edge genes). Forty-five genes were common to both sets, representing genes that share similar smoking-related expression patterns across all three airway epithelial tissues. This analysis demonstrates a common response to cigarette smoke exposure in cells lining the respiratory tract. Within this set are multiple genes involved in each of several processes including detoxification, cell cycle progression and cell adhesion. In addition, a common set of genes was down-regulated in response to smoking in both bronchial and nasal epithelium.
Several CYP450 genes were commonly up-regulated by smoking in only the nose and airway including CYP1A1 and CYP1B1, as well as cell cycle (CCNG2, RAB2) and cell adhesion genes (CEACAM5, CEACAM6). The presence of mutations in CYP1A1 in nasal and bronchial mucosa has been shown previously in smokers , and cytochrome P450 genes are known to be involved in xenobiotic metabolism in bronchial mucosa . Exposure of alveolar epithelial cells to environmental toxins has been shown to promote cell cycle progression , which could explain the increased expression of cell cycle genes in the nasal epithelial cells of smokers. Glutathiones such as GPX2 were up-regulated in both bronchial and buccal epithelial cells. Aldo-keto reducatse genes which are activated in response to cigarette smoke in human oral squamous cell lines , are also up-regulated in both bronchial and buccal epithelial samples. Oxidoreductase genes are up-regulated in all three airway epithelium including other CYP450 genes (CYP4F3, CYP4F11), aldehyde dehydrogenases (ALDH3A1), and aldo-keto reductases (AKR1B1), suggesting that smoking activates common detoxification pathways in exposed airway epithelial cells.
Gene set enrichment analysis also identified genes that are among the most down-regulated in both nasal and bronchial epithelium in response to tobacco exposure. SLIT2, which is a known tumor suppressor that is down-regulated in lung cancer [38, 39] is among these genes. We found HNMT, which is expressed highly in both bronchial and nasal mucosa [40, 41], and has been shown to be down-regulated with smoking in other mucosal cells , to be down regulated in smoking in bronchial and nasal epithelium. The genes that are among the most down regulated by smoking in both bronchial and nasal epithelium were enriched for those with functions in cellular localization, migration, and motility genes. These data suggest that smoking results in the down-regulation of structural genes in these tissues.
Based on the data presented here, we suggest that aspects of the bronchial gene expression response to smoking are also changed by smoking in nasal epithelium, with certain of these genes also being perturbed by smoking in buccal mucosa. This suggests that there are common features in the field of injury caused by cigarette smoke throughout the airway. Our data also suggest that the gene-expression consequences of smoking are less pronounced in buccal mucosa (see Figure 3). This could be due to a number of factors: 1) the effects of smoking on buccal mucosa might indeed be less pronounced; 2) there may be more inter-subject variability in buccal mucosa gene expression; 3) the partial degradation of the RNA in the buccal mucosa samples may contribute to variability in gene expression estimates. Due to the high concentration of RNAses found in saliva, RNA obtained from buccal epithelial cells was subject to degradation, and relatively small amounts of RNA were extracted from these cells. This required us to pool samples collected from the same individual serially over several weeks. Previous studies report similar issues with salivary RNA run on microarrays . Consistent with the low yield of partially degraded RNA from buccal samples, we detected sequence-specific hybridization intensity for fewer probesets in the buccal samples than in the nasal or bronchial samples. Despite these technical limitations, there was considerable overlap between genes that are among the most altered in response to smoking in the nose and bronchus and those that are most differentially expressed in the mouth based on the overlap between the leading edge subsets from gene set enrichment analysis. Differential expression of several genes seen to be induced by smoking in buccal mucosa was validated in buccal mucosa samples from independent volunteers using real competitive PCR. Taken together, these findings indicate that gene expression is perturbed by smoking in buccal mucosa and suggest that techniques for assaying gene expression in the context of partially degraded RNA will facilitate further studies to determine if buccal-mucosa specific factors contribute to the apparent differences in the magnitude of the smoking response of buccal mucosa relative to that seen in other airway epithelia.