Background
Tobacco is considered the single greatest cause of preventable death in the world [
1]. Chronic tobacco exposure is associated with the development of chronic lung disorders like chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, and is the main cause of lung cancer globally [
1]. A larger effect of tobacco smoke inhalation is the induction of inflammation, a process characterized by the release of soluble mediators, oxidant stress, and the recruitment of inflammatory cells into tissue [
2]. This promotes tissue remodeling, including alterations in lung structure and function, and oncogenesis [
3].
Due to nicotine’s well known addictive effects, attention has turned to this plant derived alkaloid which represents ~0.6–3.0% of the dry weight of tobacco. Recent studies have unveiled effects on lung development [
4,
5] and on inflammatory processes [
6]. This is possible because of the existence of nicotinic receptors in lung tissue capable of signal transduction [
7]. These receptors, termed nicotinic acetylcholine receptors (nAChRs), are multimeric acetylcholine-triggered channel proteins that form homomeric and heteromeric α and β chain pentameters. The α7 nAChR is the most abundant homomeric receptor form, composed of five α7 subunits. In developing primate lungs, α7 nAChRs were detected in airway epithelial cells, around large airways and blood vessels, free alveolar macrophages, alveolar type II cells, and pulmonary neuroendocrine cells [
4]. Additionally, increased α7 nAChR expression has been shown with nicotine administration in bronchial epithelial and endothelial cells as well being implicated in regulation of inflammation and cancer [
8‐
11].
Our laboratory has previously demonstrated that nicotine stimulates lung fibroblasts to express fibronectin by acting on α7 nAChRs both in vitro and in vivo [
11]. Fibronectin is a matrix glycoprotein, which is highly expressed in injured tissues, and is considered a sensitive marker of tissue injury and activation of tissue remodeling [
12,
13]. In an injured lung, fibronectin is deposited over denuded basement membranes where it is thought to support the migration of alveolar epithelial cells during repair [
13]. The excessive deposition of fibronectin, however, has been hypothesized to promote disrepair [
14]. Human studies also show increased fibronectin content in the bronchoalveolar lavage fluid of smokers [
15]. However, nicotine-induced fibronectin expression and deposition are not sufficient to alter overall lung architecture and, to date, there are no data suggesting nicotine, alone, promotes significant lung tissue remodeling.
In this study, nicotine is shown to stimulate lung fibroblasts to express collagen type I. This fibrillar collagen is another extracellular matrix component highly expressed in tissues during injury and repair, and its expression signals activation of tissue remodeling. Collagen type I is the most abundant form of collagen in the human body and is present in connective tissue throughout the body including tendon, ligament, skin, and lung tissue. Each rope-like procollagen molecule is made up of three chains: two pro-α1 (I) chains, which are produced from the COL1A1 gene, and one pro-α2 (I) chain, which are produced from the COL1A2 gene. After processing, the resulting mature collagen molecules arrange themselves into long, thin fibrils. Individual collagen molecules are then cross-linked to one another within these fibrils thereby forming strong collagen fibrils. Studies performed in vivo confirmed nicotine induction of collagen type I without changes in overall lung architecture in lung matrix. Also, we found that nicotine-treated fibroblasts produce a collagen-containing matrix capable of stimulating monocytic cells to produce the pro-inflammatory cytokine IL-1β in vitro.
Together, these observations suggest that nicotine stimulates alterations in the relative composition of the lung extracellular matrix favoring fibronectin [
11] and collagen type I (this report) expression without altering the overall tissue architecture of the lung. These subtle changes may render the host susceptible to excessive tissue damage after injury.
Discussion
Tobacco-related lung disease is an important health problem worldwide. Several studies suggest that tobacco exposure promotes lung tissue remodeling through oxidant stress, inflammation, and the induction of matrix-degrading proteases, among other mechanisms. The latter is supported by animal studies showing that overexpression of matrix metalloproteinase (MMP)-1 promotes the development of emphysema in transgenic mice, while the lack of MMP-12 is protective [
25,
26]. Furthermore, alterations in MMPs and other proteases have been detected in humans with tobacco-related lung disease [
27]. Unfortunately, this information has not yet translated into the development of effective therapeutic strategies. Here, we explore another mechanism of action, the induction of lung tissue remodeling through stimulation of extracellular matrix deposition. We hypothesized that nicotine, a major component of tobacco, is not only involved in tobacco addiction, but stimulates lung fibroblasts to release matrix components that affect the relative composition of the lung matrix. Consistent with this idea, we found that nicotine stimulates lung fibroblast expression and release of collagen type I up to 72 h.
Previously, we reported that nicotine stimulates the expression in lung of fibronectin, a matrix glycoprotein implicated in injury and repair [
11]. However, fibronectin matrices are often considered ‘
transitional’ matrices whose deposition does not necessarily lead to irreversible changes in tissue architecture in the absence of other factors. The discovery that nicotine also stimulates the deposition of fibrillar collagens (this report) is important because it suggests that the effects of nicotine on matrix composition may be more permanent. Collagen type I is highly expressed in injured lungs as demonstrated in acute lung injury, COPD, and chronic fibrotic lung disorders [
28]. Additionally, collagen has been associated with extralobar pulmonary artery stiffening caused by chronic hypoxia [
29], induction of epithelial-to-mesenchymal transition in non-small cell lung cancer cell lines [
30], and stimulation of cell chemotaxis by its fragments [
31].
Considering the importance of the proposed mechanisms of action, we turned our attention to the pathways involved in stimulation of collagen expression. We found that nicotine affected collagen expression in lung fibroblasts by acting on α7 nAChRs. Several studies have implicated α7 nAChRs in lung branching morphogenesis and in the pathogenesis of lung cancer [
32,
33]. More recently, α7 nAChRs were found to mediate the effects of nicotine in developing lungs [
5,
34]. Specifically, morphological airway abnormalities and airflow limitation were detected in the offspring of nicotine-treated wildtype animals, but not in animals lacking α7 nAChRs. Interestingly, and reminiscent to our work, collagen was found to be upregulated around the airways of animals exposed to nicotine. Based on the information presented here and the growing number of publications implicating α7 nAChRs in several disease states, it is reasonable to consider α7 nAChRs as promising targets for drug development to counteract the deleterious effects of tobacco. This is now possible considering that the technology to develop safe and effective agents that target nAChRs are currently available for human use [
35].
Another important finding was that nicotine also stimulated fibroblast proliferation, a process capable of further promoting tissue remodeling. This effect was also mediated via α7 nAChRs as demonstrated by the lack of response in cells silenced for α7 nAChRs. In prior work, we and others demonstrated that nicotine leads to ERK activation [
11]. Consequently, we tested the role of ERK and found that a MEK-1/ERK inhibitor, PD98059, inhibited nicotine-induced fibroblast proliferation.
To determine the potential relevance of our findings, we tested nicotine exposure in vivo. Previous studies have shown nicotine increases collagen type I expression in vivo in rhesus monkeys [
36]. We exposed mice to nicotine in their drinking water for 8-12 weeks. When examined, the harvested lungs from nicotine-treated wildtype mice showed increased collagen deposition predominating around airway and vascular structures as determined by immunohistochemistry. Consistent with our in vitro findings, lung tissue also showed increased phosphorylation of ERK. We also detected increased phosphorylation of Smad3, a transcription factor known for mediating many of the pro-fibrotic effects of transforming growth factor-β. Increased Smad3 has been associated with collagen expression in cardiac and dermal fibroblasts [
37,
38].
Our findings suggest that nicotine can promote fibronectin [
11] and collagen type I (this report) deposition in lung without affecting the organ’s overall architecture. We refer to this process, which appears to be self-limiting, as “transitional remodeling”. Since deposition of new collagen fibrils in our model was not associated with dramatic alterations in lung architecture, how then do these subtle changes in lung matrix composition affect the lung? We reasoned that newly deposited collagen fibrils do not affect the lung in the absence of other injurious stimuli, but instead, may influence immune cell function after injury. Consistent with this idea, our lab has previously shown that purified collagen type I can robustly activate monocytic IL-1β expression [
39]. In this report, we show that collagen-containing matrices derived from nicotine-treated fibroblasts are capable of activating monocytic cells and stimulating their expression of the pro-inflammatory cytokine IL-1β. This pro-inflammatory event was inhibited by pretreatment of fibroblasts with antibodies against α2β1, a collagen-binding integrin [
40], PD98059 (MEK-1 inhibitor), MG 624 (α7 nAChR inhibitor), or matrices derived from nicotine-treated α7KO primary lung fibroblasts when compared to control. In vivo, increased IL-1β gene expression and staining was also detected in the lungs of nicotine-treated mice (Fig.
5e and f). The increased IL-1β staining detected in nicotine-treated lungs is likely on macrophages, which nicotine has previously been shown to activate [
41]. This suggests that nicotine has the capacity to activate resident macrophages in addition to recruiting circulating macrophages. Integrins also control immune responses in T cells. For example, integrin-mediated binding to collagen provides a co-stimulatory signal for T cell activation [
42], resulting in increased proliferation and secretion of pro-inflammatory cytokines such as TNF-α and IFN-γ [
43]. Thus, by promoting subtle alterations in matrix composition, nicotine may indirectly stimulate the exaggerated expression of pro-inflammatory cytokines (e.g., IL-1β) by immune cells recruited to the lung after injury, thereby helping perpetuate inflammation, a process considered important in the pathogenesis of tobacco-related lung disorders.
Elements of lung transitional remodeling have also been demonstrated in alcohol-exposed rats and mice [
17,
44], alcoholic subjects [
45], post-lung transplant recipients [
46], and aging mice [
14]. However, the implications of lung transitional remodeling are unknown. It is presumed that if the stimulating agent is eliminated, a ‘normal’ matrix is restored. In contrast, persistence of the transitional matrix may lead to ineffective repair after injury through the induction of pro-inflammatory agents directly or via the release of matrix fragments [
47]. We and others have suggested that these changes may explain the increased incidence and mortality observed for acute lung injury in alcoholics [
20,
45], the predisposition to lung cancer in smokers [
8,
48], the development of rejection after lung transplantation [
46], and the worse outcomes observed in elderly patients with pulmonary disorders [
14].
Additionally, it is important that we emphasize the implications of this research to understanding the potential impact (and safety) of e-cigarettes, an area that remains relatively unexplored. Consistent with this report, early studies suggested that e-cigarettes cause similar cell changes as those caused by tobacco exposure [
49]. Considering this, in addition to serious concerns about their ability to serve as a gateway drug for smoking and current non-smokers, the use of these agents might pose a serious setback for global health with the use of e-cigarettes in adolescents, which has doubled yearly [
50,
51].
However, until further studies are performed, these statements remain highly speculative. Booth et al., recently published a technique to isolate acellular lungs, which could provide the methods to study this interaction in a ‘
transitional matrix’ lung [
52]. Nevertheless, the idea that lung transitional remodeling may precede processes such as COPD, acute lung injury and pulmonary fibrosis, among other disorders, is tantalizing and testable.