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Nicotine and fibronectin expression in lung fibroblasts: implications for tobacco-related lung tissue remodeling

JESSE ROMAN^*,1, JEFFREY D. RITZENTHALER^*, ALCIDES GIL-ACOSTA^*, HILDA N. RIVERA and SUSANNE ROSER-PAGE

^* Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine; and
Atlanta Veterans Affairs Medical Center, Atlanta, Georgia, USA

¹Correspondence: Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Whitehead Biomedical Research Building, 615 Michael St., Suite 205-M, Atlanta, GA 30322, USA. E-mail: jroman{at}emory.edu

SPECIFIC AIMS

Tobacco-related lung diseases such as emphysema and chronic bronchitis have been linked to excess deposition of connective tissue molecules such as the matrix glycoprotein fibronectin. The objective of this work is to explore intracellular mechanisms by which nicotine, a major component of tobacco, stimulates expression of fibronectin by lung fibroblasts both in vitro and in vivo.

PRINCIPAL FINDINGS

1. Nicotine stimulates expression of fibronectin in cultured primary and transformed lung fibroblasts
To examine the effects of nicotine on fibronectin expression, primary rat tracheal and murine lung fibroblasts, IMR90 fetal lung fibroblasts, and NIH 3T3 fibroblasts were exposed to nicotine in vitro. Endogenous mRNA levels for fibronectin were measured using RT-PCR. Nicotine induced the accumulation of fibronectin mRNA levels in all fibroblasts tested in a dose- and time-dependent manner. Stimulation of fibronectin mRNA expression by nicotine was associated with an increase in production of fibronectin protein as determined by Western blot. To examine the role of nicotine on fibronectin gene transcription, we studied NIH 3T3 fibroblasts stably transfected with a DNA construct containing 1.2 kb of human fibronectin promoter fused to a luciferase reporter gene, pFN(1.2kb)LUC. Nicotine induced transcription of the fibronectin gene.

2. Nicotine-induction of fibronectin is dependent on protein kinase activation
To test the role of protein kinases in nicotine-induced fibronectin expression, fibroblasts were exposed to nicotine (50 µg/mL) and tested first for protein kinase C. cPKC protein increased after 30 min of exposure to nicotine (Fig. 1 A). To evaluate the role of cPKC, two potent inhibitors of PKC activation, calphostin C and chelerythrine chloride, were tested on fibronectin gene transcription. Both light-activated calphostin C (1x10^–7 M) and chelerythrine chloride (1 µM) ablated expression of fibronectin, whereas inactive calphostin C had no effect (Fig. 1B, C ). We tested the potential role of mitogen-activated protein kinases, or more specifically ERK-1 and ERK-2, in the nicotine-induced fibronectin pathway. Nicotine not only increased expression of ERK-1/ERK-2 protein, but also increased the activated forms (p-ERK-1/p-ERK-2) as determined by Western blot (Fig. 1D ). Increase in ERKs was also associated with increased detection of activated MAPK as early as 2.5 min after stimulation with nicotine as determined using an in-gel kinase assay (Fig. 1E ). To confirm a role for MAP kinases in the nicotine-induced fibronectin response, we tested effects of the inhibitor PD98059 (50 µM), a highly selective inhibitor of MEK-1 activation and the MAP kinase cascade that leads to induction of ERKs. PD98059 abrogated nicotine-induced fibronectin gene transcription (Fig. 1F ). Finally, we found that nicotine also induced intracellular accumulation of cyclic AMP as determined using a direct cyclic AMP enzyme immunoassay. This observation, together with the increase in fibronectin gene transcription observed in fibroblasts treated with dbcAMP (an analog of cyclic AMP) and forskolin (an inducer of adenyl cyclase), suggests a role for cyclic AMP-dependent kinases in nicotine induction of fibronectin.

View larger version (18K): [in this window] [in a new window]   Figure 1. Nicotine induces fibronectin expression via protein kinase-dependent pathways. A) NIH/3T3 fibroblasts were cultured in the presence of nicotine (50 µg/mL) for 15 and 30 min followed by Western blot to detect cPKC. An increase in cPKC protein was detected at 30 min. B, C). Transfected cells were cultured in the presence of nicotine (50 µg/mL) with or without active calphostin C (ACC, 1 x 10–7 M) or inactive calphostin C (ICC) or chelerythrine chloride (1 µM) for 24 h. Cells were harvested and fibronectin gene transcription was measured by luminescence. Active calphostin C (ACC) and chelerythrine chloride (CHE) inhibited nicotine-stimulated expression of the fibronectin gene. Inactive calphostin C (ICC) had no effect. D) NIH/3T3 fibroblasts were cultured in the presence of nicotine (50 µg/mL) for 24 h, harvested, and processed for Western blot to detect ERK-1, ERK-2, and phosphorylated ERK-1, ERK-2. Nicotine induced expression of both ERK-1/ERK-2 and phosphorylated ERK-1/ERK-2 proteins (each tested in duplicate). E) NIH/3T3 fibroblasts were treated with nicotine for up to 60 min as described above and submitted to an in-gel kinase assay to detect activated MAP kinase. Note that nicotine induced MAP kinase activation by 2.5 min. F) Transfected fibroblasts were cultured in the presence or absence of MEK-1 inhibitor PD98059 (50 µM) for 24 h. Afterwards, fibronectin gene transcription was measured by luminescence. Note that MEK-1 inhibitor prevented the stimulatory effect of nicotine (P<0.02). Results are depicted as mean ± SD. GAPDH was used to control for loading for all Western blots.

3. Nuclear binding factor CREB is involved in nicotine-induced transcription of the fibronectin gene
We and others have shown that CREB phosphorylation is important for stimulation of fibronectin expression. To study the role of CREB, lung fibroblasts were treated with nicotine and the amount of phosphorylated CREB nuclear binding factor was measured using Western blot analysis. An increase in phosphorylated/activated CREB protein was detected as early as 2 h after nicotine treatment and peaked at 4 h. Increase DNA binding by CREB was demonstrated by electrophoretic mobility gel shift assays in fibroblasts treated with nicotine when compared with nontreated control cells. The role of CREB in nicotine-induced fibronectin gene transcription was confirmed in cotransfection studies where fibroblasts were transfected with pFN(1.2kb)LUC. Cotransfection with a consensus CREB competing oligonucleotide was able to abrogate the nicotine-induced increase in fibronectin gene transcription. CREB affects fibronectin gene transcription by interacting with cyclic AMP response elements (CRE) in its promoter. Using cells transfected with deletion mutant DNA constructs, we observed that stimulation of fibronectin gene transcription by nicotine was greatly diminished in mutants lacking CRE sites.

4. Nicotine-induced fibronectin response is mediated by nicotinic acetylcholine receptors (nAChRs)
Nicotine has been shown to act via cholinergic pentameric channels termed nicotinic acetylcholine receptors (nAChRs). Consistent with a role for nAChRs in the nicotine-induced fibronectin response, we found that acetylcholine mimicked the effect of nicotine on fibronectin expression, whereas atropine inhibited it. However, since these reagents are not specific for nAChRs, we tested effects of –bungarotoxin (-BGT), a known inhibitor of 7 nAChRs. -BGT (10 nM) prevented nicotine-induced expression of the fibronectin gene (Fig. 2 A). Mecamylamine, which does not affect 7, but does modify binding to other nAChRs (e.g., 3), did not block the nicotine-induced response. Nicotine induced 7 nAChR mRNA in NIH/3T3, IMR90, primary rat tracheal, and primary mouse lung fibroblasts (Fig. 2B ). In addition, nicotine treatment caused a significant increase in the binding of ¹²⁵I--bungarotoxin when compared with nontreated control cells. To test the specificity of -bungarotoxin binding, unlabeled -bungarotoxin or nicotine was added to the media and ¹²⁵I--bungarotoxin binding was measured. Both unlabeled -bungarotoxin and nicotine alone were able to block nicotine-induced ¹²⁵I--bungarotoxin binding.

View larger version (22K): [in this window] [in a new window]   Figure 2. Role of nicotinic acetylcholine receptors in nicotine-induced fibronectin response. A) Transfected NIH/3T3 fibroblasts were treated with nicotine (50 µg/mL) for 24 h in the presence or absence of -bungarotoxin (10 nM). Cells were harvested and fibronectin gene transcription was measured by luminescence. -bungarotoxin completely abolished induction of fibronectin by nicotine. B) NIH/3T3 fibroblasts, human lung fibroblasts (IMR-90), and primary rat tracheal and murine lung fibroblasts were exposed to nicotine as described before for 0–24 h. Cells were harvested and processed for RT-PCR to detect 7 nAChR mRNA. 7 mRNA was detected in all fibroblasts studied and nicotine induced accumulation of 7 mRNA.

5. Nicotine induces fibronectin expression in vivo
C57BL/6 mice were provided nicotine (100 µg/mL) in drinking water for 90 days. This protocol was based on published work demonstrating the effectiveness of this treatment in delivering nicotine to tissues in rodents. Lungs of these animals were harvested and tested for fibronectin mRNA using RT-PCR, and for fibronectin protein by Western blotting and immunohistochemistry. We observed increased expression of fibronectin mRNA in lungs of animals exposed to nicotine when compared with controls. Immunohistochemical analysis revealed that nicotine exposure was associated with increased fibronectin protein in alveolar septae as well as in airway epithelial cells and vascular structures.

CONCLUSIONS AND SIGNIFICANCE

This report establishes a link between nicotine exposure and fibronectin expression in lung fibroblasts both in vitro and in vivo and has three key findings (Fig. 3 ). First, the report delineates the intracellular signaling pathways involved in fibronectin induction by nicotine in fibroblasts. It shows that induction of fibronectin gene transcription is dependent on the activation of protein kinase C and mitogen-activated protein kinases, the intracellular accumulation of cyclic AMP, and the phosphorylation and DNA-binding by transcription factor CREB. Second, the work suggests that members of the pentameric cholinergic nAChR family mediate these events. Our data suggest that 7 nAChRs mediate stimulation of fibronectin by nicotine in fibroblasts. This and related investigations are beginning to unveil novel roles for nAChRs in lung tissue remodeling. Finally, we demonstrated induction of fibronectin mRNA and protein in lungs of mice exposed to nicotine in drinking water, suggesting that our observations in vitro are likely to be relevant to the situation in vivo.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized link between nicotine exposure and fibronectin expression in lung fibroblasts. 7 nAChRs on the surface of fibroblasts mediate stimulation of fibronectin by nicotine. Induction of fibronectin gene transcription is dependent upon activation of protein kinase C, mitogen-activated protein kinases, intracellular accumulation of cyclic AMP, and the phosphorylation and DNA binding of the transcription factor CREB.

This work has prompted us to postulate that nicotine can induce exaggerated expression and deposition of fibronectin in lungs of animals exposed to tobacco. This, in turn, is likely to affect the overall composition of lung extracellular matrix. In doing so, nicotine promotes tissue remodeling around airways and within lung parenchyma, and this is likely to represent an important mechanism by which tobacco results in abnormal lung function.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0826fje; doi: 10.1096/fj.03-0826fje

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