Cigarette smoking is the major environmental exposure that leads to the pathogenesis of COPD [
1‐
5]. The oxidative stress that cigarette smoke places on the airway epithelium results in a series of predictable morphologic changes over time. Despite our understanding of the pathologic changes that occur in the airways, the molecular mechanisms that direct the response of the airway epithelium to cigarette smoke are only partially understood [
1‐
5]. The present study on pirin emerged from our laboratory's ongoing effort to identify unique cellular pathways that are linked to the development of lung diseases, such as COPD and lung cancer, by using gene expression analysis of the airway epithelium of smokers.
Apoptosis of airway epithelial cells is relevant to the pathogenesis of chronic bronchitis because disruption of epithelial integrity in the central airways appears to be an early event in response to cigarette smoke [
6‐
8,
32]. Based on the hypothesis that there may be apoptosis-related proteins expressed in the epithelial cells of cigarette smokers that have not been identified, we employed the unbiased strategy of assessing gene expression in airway epithelial cells of smokers
vs non-smokers for the up-regulation of candidate proteins using microarray technology. Affymetrix gene chip assessment of airway epithelial cells from phenotypically normal ~20 pack-yr smokers compared to normal non-smokers demonstrated the up-regulation of several proteins that have been linked to apoptosis in the airway epithelium of smokers compared to non-smokers. Among the up-regulated proteins that were found to be associated with apoptosis was pirin, a nuclear transcription cofactor that is part of the cupin superfamily [
21,
33,
34].
TaqMan real-time RT-PCR independently confirmed the microarray observed changes in pirin mRNA expression. To validate this in vivo observation, an in vitro model was designed to test the effect of cigarette smoke on pirin expression in epithelial cell culture. Using primary cultures of human bronchial epithelial cells, pirin expression was shown to respond to increasing concentrations of cigarette smoke extract relatively acutely, within 24 to 48 hr. A similar increase in pirin RNA expression level was observed in the BEAS-2B cell line when exposed to cigarette smoke extract. The potential for pirin to induce apoptosis in bronchial epithelial cells was assessed by using AdPirin, an adenovirus gene transfer vector expressing the human pirin cDNA, to over-express pirin in cultures of transformed airway epithelial cells. The data demonstrate that pirin over-expression results in significant epithelial cell apoptosis, a possible mechanism that leads to a breakdown of the epithelium in response to cigarette smoke. Although the magnitude of pirin over-expression is greater in the in vitro AdPirin experiments than that observed in vivo, it is important to recognize that the in vivo upregulation of pirin expression occurs after chronic smoking (~20 pack-yr), and this chronic, low level up-regulation over an extended period of time is impossible to emulate in vitro. It is therefore not surprising, that in order to demonstrate an in vitro effect on apoptosis over a much shorter time course (2–3 days), a higher expression of the gene was required. Interestingly, in the experiments where bronchial epithelial cells (BEAS-2B) were exposed to cigarette smoke extract, there was a similar 2 fold increase in pirin expression to 10% CSE and a significant increase in apoptosis, thus paralleling the in vivo observation.
Apoptosis and the Pathogenesis of Chronic Bronchitis
One of the main functions of the airway epithelium is to provide a physical barrier to protect the underlying tissue from the harmful effects of cigarette smoke [
1,
2,
4,
5]. The inflammatory response seen in COPD is initiated when the airway epithelium is disrupted by chronic exposure to cigarette smoke [
4]. This breach in epithelial integrity triggers the innate immune response which mobilizes neutrophils, eosinophils, macrophages, natural killer cells and mast cells into the subepithelial space [
2,
9]. When the stress of cigarette smoke is exerted over years, the persistent inflammation in the central airways leads to the pathologic finding of chronic bronchitis. As the inflammation extends to the small airways (between 4
th to 14
th generation bronchi), inflammatory exudates can accumulate in the lumen of the bronchi resulting in airflow obstruction [
9].
Epithelial permeability, as measured by
99mTC-DTPA lung clearance in human subjects, has been shown to be increased in chronic smokers compared to non-smokers [
7,
32]. Studies using animal models have shown that epithelial cell permeability can increase in response to cigarette smoke with 30 min and 6 hr of exposure [
8,
11]. This is followed by an increased number of neutrophils and mast cells in the airway wall 6 hr after cigarette smoke exposure. The mechanism for increased permeability is unclear, but prior studies have shown that damage to the epithelial cell itself, and to a lesser extent, disruption in the tight junctions between epithelial cells are partly responsible [
10,
11]. Consistent with the bronchial epithelial cell studies,
in vitro cellular studies have shown that alveolar epithelial cell viability decreases after exposure to cigarette smoke in a time and concentration dependent manner [
35].
The mechanism of cell death for the epithelium in response to cigarette smoke may involve some necrosis, but apoptosis is likely the dominant mechanism [
18]. Mouse models of smoking induced airway disease have demonstrated that apoptosis can occur in the airways in response to cigarette smoke [
16,
17]. Due to the efficient nature of the apoptotic pathway, allowing for rapid clearance of dying cells, it can be technically difficult to assess for apoptosis from
in vivo studies of the airway in humans.
Several
in vitro studies have shown that apoptosis can occur in response to cigarette smoke extract (CSE) in a variety of cell types, including airway epithelial cells, alveolar macrophages, endothelial cells, alveolar epithelial cells, and fibroblasts [
13‐
15]. In contrast, Wickenden et al [
19] found that CSE induced necrosis of a variety of cell types, while inhibiting the activation of the caspase system and preventing apoptosis. This discrepancy may be explained by the observation that at low concentrations, CSE induces apoptosis, while at higher concentrations, necrosis is induced [
18]. Liu et al [
20] reported that CSE caused DNA damage and initiated DNA repair processes, but not apoptosis, in bronchial epithelial (BEAS-2B) cells. The reason their conclusions differ from ours is likely due to differences in the concentration of cigarette smoke extract used. The maximum concentration that Liu [
20] et al used was 10% CSE generated from 1 cigarette bubbled into sterile water. Similarly, in our experiment we did not observe apoptosis in BEAS-2B cells exposed to 10% CSE, but did observe apoptosis in cells exposed to 50% CSE, generated from 4 cigarettes bubbled into LHC-9 media. Taking all of the
in vitro studies into consideration, one potential explanation is that at low concentrations, CSE causes DNA damage that is reparable until a threshhold concentration is reached, after which the cell initiates apoptosis. Very high levels of CSE likely cause overt necrosis.
Induction of Apoptosis by Pirin
Pirin was first identified by Wendler et al [
33], where it was found to interact with nuclear factor 1/CCAAT box transcription factor (NF1/CTF1). Pirin was found to be exclusively nuclear, expressed in most tissues and highly conserved among mammals. The N-terminus shares significant homologies with proteins from plants, fungi and prokaryotes.
Pirin was later found to bind to the oncoprotein B-cell lymphoma 3-encoded (Bcl-3), a member of the IκB family[
36]. Bcl-3 interacts with the anti-apoptotic protein NF-κB, by binding to the p50 and p52 [
36]. Recently, the crystal structure of pirin has been elucidated and was found to have a single Fe
2+ ion in the highly conserved N-terminal region [
34]. This metal ion may be necessary for its interaction with Bcl-3 and NF-κB. Bcl-3 can either inhibit or enhance NF-κB DNA binding and transcription depending on its binding to other transcription co-factors.
Pirin stabilizes the formation of quaternary complexes between Bcl-3, NF-κB, and its DNA target. While it is not clear exactly how pirin influences the interaction between Bcl-3 and NF-κB, based on our results and others, it seems possible that pirin directs NF-κB DNA binding towards a pro-apoptotic response. Elucidating this mechanism will require further investigation to understanding the signaling that results from pirin expression. This may not be possible through gene expression studies alone, since several apoptotic pathways are induced by mechanisms other than up-regulation of mRNA levels of pro-apoptotic genes. This might explain why there was no observed upregulation of traditional apoptotic or inflammatory pathway genes, e.g., caspases and Bcl-2, in our microarray data obtained from human airway epithelium. The lack of up-regulation of pro-inflammatory genes in the airway epithelium of smokers is also supported by the transcriptome analysis of Spira et al [
37]. One possible explanation for this observation is that many of these inflammatory mediators may be released primarily by non-epithelial cells.
Evidence for pirin involvement in apoptosis comes from experiments in tomatoes [
21]. When tomato cells in suspension are treated with the topoisomerase inhibitor, campothecin, a potent initiator of cell death,
le-pirin, an ortholog to human pirin, mRNA levels are dramatically increased. In the cyanobacterium
Synechocystis, pirin orthologs are highly induced under conditions of severe salt stress [
28].
We originally noted pirin to be up-regulated in microarrays of phenotypically normal individuals that smoke [
24], a finding also made by Spira et al [
37], in their study of genes up-regulated in the airway epithelium of smokers. Interestingly, Spira et al [
37] observed that the pirin levels returned to normal for ex-smokers, supporting observation in the present study that pirin is influenced by acute exposure to cigarette smoke.