Background
Attachment and migration of airway epithelial cells is an important aspect of repair of injury induced by allergens and other agents in asthma. Cytoskeletal reorganization, mediated through actin filament remodeling, is required to facilitate attachment and migration of epithelial cells into damaged areas. The vasodilator-stimulated phosphoprotein (VASP) binds to profilin, F- and G-actin, and mediates actin assembly, bundling and attachment at focal adhesions [
1,
2]. VASP and two other members of the ENA/VASP family in mammalian cells, MENA (mammalian ENAbled) and EVL (ENA/VASP-like) slow or inhibit fibroblast motility by altering the dynamics of actin filament structure [
3,
4]. However, MENA and EVL, despite shared molecular similarities and normal expression, did not substitute for VASP and reverse compromised cell motility in a wound healing assay of VASP-/- fibroblasts [
5].
VASP phosphorylation occurs on two serine and one threonine (Ser 157, Ser 239 and Thr 278) through cAMP and cGMP dependent protein kinases A and G [
6‐
9], and as recently observed in smooth muscle cells, protein kinase C [
10]. Phosphorylation by cAMP protein kinase preferentially occurs at Ser 157, whereas phosphorylation by cGMP protein kinase preferentially occurs at Ser 239, both kinases secondarily acting on Thr 278 [
7]. The Ser 157 phosphorylation results in decreased migration of the VASP molecule from 46 KD to 50 KD in SDS-gel electrophoresis [
6‐
9]. Functionally, phosphorylation of VASP regulates its interaction with actin, and correlates with detachment and spreading of fibroblasts and epithelial cells [
3,
4,
9,
11,
12]. Thus, the ratio of the 50 KD to the 46 KD molecular form of VASP within a cell sample provides a measure of VASP phosphorylation and indicates active remodeling of the actin cytoskeleton during cell attachment and migration.
We postulated that repair of injury to the airway epithelium during antigen-induced inflammation would involve enhanced phosphorylation of VASP in order to mediate actin cytoskeleton remodeling and cell migration into damaged areas. We further hypothesized that inhalation of the β-agonist albuterol, would also increase phosphorylation of VASP by stimulation of cAMP-dependent protein kinase activity, and result in altered epithelial cell attachment and mobility. These hypotheses were tested in three protocols using brush samples of airway epithelium obtained from allergic asthmatics and control subjects.
Discussion
Recent investigations into asthma pathogenesis have begun to focus, not on the acute inflammatory events involved with asthmatic airway inflammation, but on aberrant repair mechanisms which appear to be present [
28‐
30]. This work focused on vasodilator-stimulated phosphoprotein (VASP) which is predicted to be involved in epithelial repair mechanisms by mediating focal adhesion, actin filament binding and polymerization, and ultimately, epithelial cell mobility. Reports published during the course of our studies have shown that detachment of kidney epithelial cells increases protein kinase A activity and its phosphorylation of VASP [
12], as well as a transient increase in VASP expression [
31]. Although there was some increase in total VASP (46 KD + 50 KD forms) in asthmatics compared to normals on day 2 in the recovery from injury protocol, the difference was not significant, and probably does not contribute substantially to cell adherence and motility. We hypothesized that asthmatics would demonstrate increased VASP phosphorylation compared to nonasthmatic, normal subjects to permit actin remodeling and cell migration for repair of inflammatory injury. Instead, we observed a potential defect as shown by decreased VASP phosphorylation in asthmatic epithelial cells prior to and following allergen-induced injury compared to normal subjects. Increased VASP phosphorylation in response to segmental antigen challenge was modest in both asthmatic and nonasthmatic normal epithelial cells, but overall a profound decrease in the VASP phosphorylation was observed in asthmatic epithelial cells at all time points examined. The reduced VASP phosphorylation in epithelial cells of asthmatics was confirmed by expanded examination of an additional group of subjects, both asthmatic and normal (Table
4). It has been previously shown that blocking of phosphorylation at MENA Ser 236, which corresponds in molecular structure to VASP Ser 157, or depletion of ENA/VASP results in increased cell spreading and reduced functional control of cell motility in a number of model systems [
3‐
5,
11]. More directly, 10 μM PGE
1 converts 60% of the 46 KD VASP to 50 KD VASP and completely inhibits platelet aggregation as a measure of cell-cell adhesion [
6]. Thus, even partial conversion of VASP to its phosphorylated form has significant impact. This suggests one potential mechanism for aberrant epithelial repair in asthmatics: defective or diminished VASP phosphorylation may indicate abnormal epithelial motility. Confirming defective epithelial cell motility in asthmatics
in vivo will be challenging, but is a necessary next step in this work.
While segmental antigen challenge did not significantly increase VASP phosphorylation in the recovery from injury protocol "a", there was a trend toward a significant increase, which was confirmed in asthmatics enrolled in the regular β-agonist inhalation protocol "b". In addition, leukocytes in bronchoalveolar lavage fluid from asthmatics co-cultured with autologous epithelium did significantly increase VASP phosphorylation, unlike the leukocytes from nonasthmatic normal subjects, suggesting a soluble signal from the BAL leukocytes to epithelial cells in asthmatics. What the signal(s) may be remains to be determined, but other work from our laboratory suggests certain growth factors such as TGFβ, could be involved.
Because VASP becomes phosphorylated by cAMP-dependent protein kinase, we also hypothesized that both
in vivo and
in vitro exposure of epithelial cells to a β-agonist, which increases cyclic AMP levels, should increase VASP phosphorylation. As predicted, regularly inhaled albuterol increased VASP phosphorylation
in vivo and apparently altered epithelial cell adhesion, producing significantly greater numbers of epithelial cells shed into bronchoalveolar lavage fluid from unchallenged lung segments exposed only to the β-agonist, without any allergen-induced inflammation. The β-agonist effect on VASP phosphorylation was short-lived and the ratio of 50/46 KD VASP returned to baseline within 12 hr of the last albuterol inhalation in vivo. Whether the increased epithelial cell detachment induced by albuterol inhalation returns to baseline as quickly has not been determined. β-agonist use may therefore produce conflicting effects on asthmatic epithelium. β-agonists inhibit keratinocyte migration by β
2-adrenergic receptor activation of the serine/threonine phosphatase PP2A [
32], the principal phosphatase which dephosphorylates VASP [
33], and at the same time activate cAMP-dependent protein kinase phosphorylation of VASP [
6‐
9]. Thus, β-agonists may promote epithelial repair by enhancing both phosphorylation and dephosphorylation of VASP in actin filament restructuring, but may also promote epithelial damage by increasing the detachment of epithelial cells from the airway. These results also caution against considering as "baseline" or "control," samples obtained from asthmatics receiving β-agonist therapy without an appropriate washout interval.
Genetic variation and altered function of the β2-adrenergic receptor could potentially contribute to the differences in VASP phosphorylation observed between asthmatics and nonasthmatic normal subjects [
27]. However, examination of 50/46 KD VASP ratio grouped according to β2-adrenergic receptor haplotype for our study subjects did not reveal any specific haplotype bias, either homozygous or heterozygous, affecting VASP phosphorylation. Although the numbers of subjects in any one haplotype classification were limited, the largest 2/4 haplotype group, nonasthmatic normal subjects showed a significantly increased ratio compared to asthmatic subjects. It is possible that other characteristics of the β2-adrenergic receptor gene (for example, stability of message due to variation in the 3' untranslated region, [
34]), variation in other components in the signaling cascade such as cAMP dependent protein kinase A [
6‐
9], or activation of protein kinase C [
10], genetic variation in VASP itself, or differences in the activity of protein phosphatases [
33] between asthmatics and normals may contribute to the observed disparity in VASP phosphorylation here between asthmatic and normal subjects. Work is currently addressing these areas of inquiry.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
ATH conceived the study, supervised data collection, statistical analysis and interpretation, and drafted the manuscript. MW and KAR processed samples from bronchoscopy, and analyzed VASP data from western blots. GCF processed DNA from subjects, determined β-adrenergic receptor haplotype, and with GAH, sequence of β-adrenergic receptor; both provided drafting and critical comment on revision of the manuscript. RC, VB and JGZ consented and enrolled subjects, performed bronchoscopies, processed samples and critically commented on the manuscript. SPP participated in study design and coordination, analysis, writing and critical revision of the manuscript.