Published ahead of print on February 22, 2007, doi:10.1165/rcmb.2006-0383OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0383OC Sphingosine-1-Phosphate/Sphingosine Kinase Pathway Is Involved in Mouse Airway HyperresponsivenessDipartimento di Farmacologia Sperimentale, Università di Napoli Federico II; Dipartimento di Medicina Sperimentale, Sezione di Farmacologia L. Donatelli, Seconda Università degli Studi di Napoli; and Dipartimento di Internistica Clinica e Sperimentale, Seconda Università di Napoli, Napoli, Italy Correspondence and requests for reprints should be addressed to Giuseppe Cirino, Ph.D., Dipartimento di Farmacologia Sperimentale, via Domenico Montesano 49, 80131 Napoli, Italy. E-mail: cirino{at}unina.it
Sphingosine-1-phosphate (S1P) has been shown to regulate numerous and diverse cell functions, including smooth muscle contraction. Here we assessed the role of S1P/Sphingosine kinase (SPK) pathway in the regulation of bronchial tone. Our objective was to determine, using an integrated pharmacologic and molecular approach, (1) the role of S1P as endogenous modulator of the bronchial tone, and (2) the linkage between S1P pathway and bronchial hyperresponsiveness. We evaluated S1P effects on isolated bronchi and whole lungs, harvested from Balb/c mice sensitized to ovalbumin (OVA) versus vehicle-treated mice, by measuring bronchial reactivity and lung resistance. We found that S1P administration on nonsensitized mouse bronchi does not cause any direct effect on bronchial tone, while a significant increase in Ach-induced contraction occurs after S1P challenge. Conversely, in OVA-sensitized mice S1P/SPK pathway triggers airway hyperesponsiveness. Indeed, S1P causes a dose-dependent contraction of isolated bronchi. Similarly, in the whole lung system S1P increased airway resistance only in OVA-sensitized mice. The action on bronchi of S1P is coupled to an enhanced expression of SPK1 and SPK2 as well as of S1P2 and S1P3 receptors. In these experiments the key role for S1P/SPK in hyperreactivity has been confirmed by pharmacologic modulation of SPKs. S1P/SPK pathway does not seem to play a major role in physiologic conditions, while it may become critical in pathologic conditions. These results open new windows for therapeutic strategies in diseases like asthma.
Key Words: S1P • sphingosine kinase • bronchus • hyperresponsiveness
Sphingosine-1-phosphate (S1P) can act as an extracellular ligand, activating specific G protein–coupled sphingolipid receptors in the plasma membrane (1, 2). Alternatively, S1P produced after activation of specific receptors can act as intracellular messenger and stimulate Ca2+ channel on the endoplasmic reticulum (3). Dissection of the relative contribute of intra- or extra-cellular S1P is difficult (4). S1P intracellular levels are regulated via the activation of the enzyme sphingosine kinase (SPK) (5–7). However, the intracellular targets have not been definitively identified. The picture is made even more complicate as S1P, by binding to its receptors, can stimulate sphingosine kinase and thus increase intracellular levels by itself (8, 9). The function of S1P has been intensely investigated over the past years, and it has become clear that S1P is involved in biological functions including cell growth and survival, differentiation, calcium homeostasis, and in many of the pathways involved in smooth muscle contraction (10–12). The emerging role of S1P in regulating smooth muscle contraction has led to exploration of the possibility that this sphingolipid metabolite could represent a potential therapeutic target. Recent studies have suggested that S1P signaling is involved in hypertension and asthma (13–16). The finding that S1P is an autocrine mediator of activated mast cells further suggests an involvement of S1P in pathophysiology of asthma and/or airway hyperresponsiveness (17, 18). An involvement of S1P in human asthma has been hypothesized on the basis that S1P levels are elevated in the airways of individuals with asthma after segmental allergen challenge (15). In addition, we have recently showed that S1P acts as a chemotactic agent for human eosinophils in vitro and in vivo (19). Pathologic changes underlying airway hyperreactivity are characterized, at least in part, by an influx of cells such as lymphocytes and eosinophils that can sustain the ongoing inflammation and provoking airway smooth muscle contraction (20–23). It has been suggested that airway smooth muscle plays a prominent role in orchestrating both the acute inflammatory reaction and the chronic processes supporting airway remodeling (24–26). The majority of studies published so far has focused on the involvement of S1P in smooth muscle hyperplasia and cytokine production (15). However, it is likely that S1P could contribute to the asthma directly through its effects on smooth muscle contractility. At the present stage the functional role of this signaling pathway in airway smooth muscle has been only partially investigated. In this study we used an integrated pharmacologic and molecular approach to determine (1) the role of S1P as endogenous modulator of the bronchial tone, and (2) the linkage between S1P pathway and bronchial hyperresponsiveness in sensitized mice.
Drugs Acetylcholine (Ach), dimethyl sulfoxide (DMSO), pertussis toxin (PTX; from Bordetella pertussis), suramine, S1P, and DL-threo-Dihydrosphingosine (DTD) were purchased from Sigma Chemical Co. (Milano, Italy). BML-241 was purchased from BIOMOL (Plymouth Meeting, PA).
Tissue Preparation
Antigen Exposure and Drug Treatment of Mice
Role of S1P in Regulation of Bronchial Tone
Isolated Perfused Mouse Lung Preparation
Western Blotting The filters were washed extensively in PBS containing 0.1% vol/vol Tween 20, before incubation for 2 h with anti–horseradish peroxidase-conjugate secondary antibody. Membranes were then washed and developed using enhanced chemiluminescence substrate (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
Statistical Analysis
Effect of S1P on Bronchial Smooth Muscle Tone Cumulative administration of S1P (1 x 10–8–3 x 10–5 mol/liter) on mouse bronchi does not cause any significant change in tone up to 10–5 mol/liter, as shown in Figure 1A. Only at the concentration of 3 x 10–5 mol/liter S1P produces a weak but not significant constrictor effect. Incubation of bronchial rings with S1P (3 x 10–7 mol/liter) before addition of Ach causes a significant increase in Ach-induced contraction (Figure 1B).
Since specific pharmacologic antagonists for S1P receptor subtypes are not commercially available for all receptor subtypes, we incubated tissues with (1) activated PTX (1 µg/ml 2 h), which blocks Gi coupling and S1P1 receptor activation; (2) suramine (100 µmol/liter, 1 h), which blocks Gs coupling and in turn S1P3 receptor activation; (3) BML-241, a specific S1P3 antagonist; and (4)Y-27632, an inhibitor of Rho kinase, since S1P2 signals through a Rho-dependent pathway (1). Rings were then incubated with S1P (3 x 10–7 mol/liter) and a cumulative concentration–response curve to Ach (10–8- 3 x 10–5 mol/liter) performed. PTX, suramine, and BML-241 do not affect S1P-induced increase in Ach-induced contraction (data not shown). Y-27632 at 10–7 mol/liter, a concentration that does not affect Ach-induced contraction, reverts the potentiating effect of S1P on Ach-induced contraction (Figure 1C). It is well known that S1P can promote, via S1Pn activation, its own intracellular synthesis through SPK pathway (8). To assess the role of this intracellular pathway on bronchial tone, bronchial preparations were incubated with DTD, a selective inhibitor of sphingosine kinase and an Ach-induced concentration curve performed in presence of either vehicle or S1P (3 x 10–6 mol/liter). Although DTD does not affect by itself Ach-induced contraction (data not shown), incubation of bronchi with DTD prevents S1P-induced potentiation of Ach contraction (Figure 1D).
Role of SPK/S1P in Mouse Airway Hyperresponsiveness
To assess the role of intracellular pathway in bronchial hyperresponsiveness, we incubated bronchial preparation with the specific sphingosine kinase inhibitor (DTD). Although DTD does not affect Ach-induced contraction in vehicle-treated mice, it significantly inhibits the hyperesponsiveness to Ach in OVA-sensitized animals (Figure 2D). Furthermore, pretreatment with DTD reverted the enhanced Ach-induced contraction in sensitized bronchi to the contractile value (expressed ad dyne/mg) displayed by bronchi harvested by control mice (Figure 2C). Similarly to what we observed in control mice, preincubation of bronchi with S1P (3 x 10–7 mol/liter) causes a significant and marked increase of Ach-induced contraction (Figure 3A). This finding further implies that S1P signaling becomes critical in pathologic conditions. To gain insight into the molecular mechanisms determining this phenomenon, we performed the same experiments operated on nonsensitized bronchi. While suramine or BML-241 weakly inhibits the modulation operated by S1P on Ach-induced contraction (Figure 3B), pretreatment of bronchi with DTD reverts this effect (Figure 3C).
To confirm that the changes seen in our experimental conditions in bronchial responsiveness to Ach were coupled to changes in the lung, RL changes to Ach were measured in anesthetized, tracheostomized, and ventilated mice using a whole-body plethysmography. RL measurements in OVA-sensitized animals show a significant airway hyperesponsiveness to Ach (Figure 4A). Similarly, while normal lung did not respond to S1P challenge, OVA-sensitized lung displayed a significant and concentration-dependent response to S1P (Figure 4B) that matched the Ach response (Figure 4A).
Modulation of S1P Receptors and Sphingosine Kinase in Sensitized Animals Functional study suggests an important role for sphingosine kinase in controlling S1P levels and thus the development of airway hyperresponsiveness. Western blot analysis confirms the up-regulation of sphingolipid pathway, showing an increased expression of S1P2 and S1P3 in bronchial tissue harvested from OVA-sensitized animals when compared with sham mice (Figure 5). Conversely, S1P1 expression was unchanged. SPK1 and SPK2 protein expression is also increased (Figure 5).
The role of SPK in the development of airway hyperresponsiveness was also investigated by administering DTD (30 µg) to mice before each administration of OVA (Day 1 and Day 7). DTD treatment in vivo significantly reduced the increased responsiveness of bronchi in vitro to S1P (Figure 6A). Moreover, pretreatment of mice with DTD reduced both the increased responsiveness to Ach (Figure 6B) and the S1P-induced potentiation of Ach-induced contraction (Figure 6C).
S1P is normally present in plasma at concentrations ranging from 0.2–0.5 µmol/liter and its level is under a tight control operated by the balance between sphingosine kinase and sphingosine phosphates. In addition, S1P can be metabolised by sphingosine lyase to phosphoethanolamine and hexadecenal (11). Growth factors, cytokines, and G protein–coupled receptor agonists can increase rapidly S1P levels upon activation of SPK (27–29). We have recently shown that there is a consistent contribution of this sphingolipid pathway through S1P in cholinergic control of vessel tone (9). It is known that cholinergic system plays an important role in asthma (30–32) and that the levels of S1P are elevated in the airways of individuals with asthma (13). Following this evidence, we have addressed the role of S1P pathway in modulation of bronchial smooth muscle tone in physiologic and pathologic conditions. Administration of S1P to mouse bronchi did not modify basal bronchial tone. At the highest concentration tested S1P caused a weak, but not significant, contractile effect, suggesting a marginal role, if any, for this lipid mediator in modulation of the bronchial tone in physiologic conditions. Thus, these data fit the concept that S1P pathway is tightly controlled in physiologic conditions. However, exposure of bronchi to S1P caused a marked increase in Ach-induced contraction. The finding that S1P per se was inactive but significantly potentiated the effect of Ach led us to hypothesize that in pathologic conditions a local increase of S1P could be an important trigger of the airway hyperresponsiveness. This hypothesis was assessed by sensitizing mice in vivo using a well-known model of allergen sensitization. When bronchi harvested from OVA-sensitized mice were stimulated with S1P, there was a dose-dependent contractile effect. In addition, we confirmed that also in OVA-sensitized bronchi incubation with an inactive dose of S1P caused an exacerbation of Ach-induced response. This result suggests that a local transient increase in S1P levels, after SPK activation, could be involved in triggering bronchial hyperreactivity. The use of SPK inhibitor in vitro confirmed this hypothesis. In fact, when either normal bronchi or OVA-sensitized bronchi were pretreated with DTD before the incubation with S1P, S1P-induced Ach potentiation was reverted. Conversely, DDT by itself was ineffective on Ach-induced contraction on normal bronchi while in OVA-sensitized bronchi caused a significant inhibition. These data strongly support our working hypothesis that SPK/S1P pathway is functionally triggered in pathologic conditions. To verify whether the in vitro hypothesis could be translated to the complex lung physiology, we performed an in vivo experiment on mice. To pursue this aim, we used a whole lung system that replicates the thorax environment in which either S1P or Ach had to travel through the lung to reach the bronchi. In these experimental conditions, S1P by itself was inactive, whereas in sensitized animals it caused an increase in airway resistance comparable to that of Ach. These data confirmed again that S1P plays a role when the airways are already activated by an inflammatory trigger. By assessing through Western blot analysis the relative protein expression of the S1P receptors we found that S1P3 and S1P2 were the most abundantly expressed. To extend to the functional study to these results, we operated a pharmacologic modulation of the response. Since specific pharmacologic antagonists to each single S1P receptor subtypes are not yet commercially available, we have incubated tissues with (1) activated PTX, which blocks Gi coupling S1P1 receptor activation; (2) suramine, which blocks Gs coupling and S1P3 receptor activation; (3) BML-241, a specific S1P3 antagoinst; and (4) Y27163, a selective Rho kinase, since S1P2 signals through a Rho-dependent pathway. PTX did not affect either S1P-induced contraction or S1P-induced increase in Ach-induced contraction, implying that Gi-sensitive extracellular receptor pathway is not involved or at least does not play a major role. Pretreatment with suramine or BML-241 abrogated S1P-induced contraction, while it did not affect S1P-induced increase in Ach-contraction. Pretreatment with the selective Rho kinase inhibitor Y-27632 abrogates S1P potentiation of Ach-induced contraction. Taken together, these data suggest that S1P is involved in bronchial function at different levels through different specific receptors. Since S1P levels are tightly regulated by SPK, we next evaluated how SPK is modulated in our experimental conditions. When we measured the levels of sphingosine kinase enzyme subtypes, we found that both isoforms SPK1 and SPK2 were up-regulated in bronchi harvested from OVA-sensitized animals. This finding well fits with the data obtained with the SPK inhibitor DTD. Indeed, DTD inhibited Ach-induced contraction only in bronchi harvested by sensitized animals, while it was ineffective in control mice. Following the above findings we asked the question whether pretreatment in vivo of mice with DTD would inhibit bronchial hyperesponsiveness in sensitized animals. Pretreatment of mice with DTD before OVA administration significantly inhibited the enhanced Ach-induced contraction in vitro. Similarly, bronchi isolated by the above-described mice showed a reduced S1P-induced contraction in vitro and did not present the phenomenon of the potentiating effect induced by S1P on Ach-induced contraction. In conclusion, our data suggest that S1P/SPK pathway seems not play a major role in physiologic conditions, since exogenous (1) S1P was ineffective in our experimental settings and (2) sphingosine kinase inhibition does not affect bronchial tone. Conversely, in pathologic conditions, such as those resembling an allergen-induced inflammation, the S1P/SPK pathway appears to play a detrimental role. It is worthy of note that S1P at concentrations well below the concentration circulating in the plasma induces a bronchial response. This indicates that a local increase of S1P in the bronchial tissue, most likely mediated through an increase in SPK expression, may mediate bronchial constriction. These findings, taken together with the fact that elevated levels of S1P have been found in BAL of patients with asthma (15), and considering the effects of S1P on cell-contributing allergen-induced lung inflammation (18, 19), suggest that this pathway may represent a novel target in designing new therapies in airway hyperresponsiveness.
* These authors have contributed equally to this work.  This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1165/rcmb.2006-0383OC on February 22, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form October 12, 2006 Accepted in final form February 8, 2007
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Abstract
CLINICAL RELEVANCE
200 µl. Every 5 min a hyperinflation (–20 cm H2O) was performed. Artificial thorax chamber pressure was measured with a differential pressure transducer (Validyne DP 45–24; Validyne Engineering, Northridge, CA) and airflow velocity with pneumotachograph tube connected to a differential pressure transducer (Validyne DP 45–15). The lungs respired humidified air. The arterial pressure was continuously monitored by means of a pressure transducer (Isotec Healthdyne Cardiovascular Inc., Philadelphia, PA), which was connected with the cannula ending in the pulmonary artery. All data were transmitted to a computer and analyzed with the Pulmodyn software (Hugo Sachs Elektronik, March Hugstetten, Germany). The data were analyzed through the following formula: P = V·C–1 + RL·dV·dt–1, where P is chamber pressure, C pulmonary compliance, V tidal volume, RL airway resistance. The airway resistance value registered was corrected for the resistance of the pneumotachometer and the tracheal cannula of 0.6 cm H2O · s · ml–1. Lungs harvested from five animals of each group (saline, OVA) were perfused and ventilated for 45 min without any treatment to obtain a baseline state. Subsequently, lungs were challenged with either Ach or S1P. Repetitive dose–response curves of Ach (from 10–8 mol/liter to 10–5 mol/liter) or S1P (from 10–7 mol/liter to 10–3 mol/liter) were administered as 50 µl bolus, followed by intervals of 15 min, in which lungs were perfused with buffer only. 
-glicerophosphate 50 mmol/liter, orthovanadate sodium 0.1 mmol/liter, Mg Cl2 2 mmol/L, EGTA 1 mmol/liter, DTT 1 mmol/liter, PMSF 1 mmol/liter, aprotinin 10 µg/ml, leupeptin 20 µmol/liter, 50 mmol/liter NaF) using a Polytron homogenizer (2 cycles of 10 s at maximum speed). After centrifugation of homogenates at 10,000 rpm for 10 min, equal amounts of the denatured proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked by incubation in PBS containing 0.1% vol/vol Tween 20 and 5% nonfat dry milk for 2 h, followed by an overnight incubation at 4°C with anti-S1P2, anti-S1P3, and anti-SPK1 or anti-SPK2. 
