| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia, USA;
* Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA and
Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia, USA
2Correspondence: Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, 2-011 Sanger Hall, 1101 E. Marshall St., Richmond, VA 23298-0614, USA. E-mail: sspiegel{at}vcu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: asthma • airway smooth muscle cells • sphingosine-1-phosphate • contractility
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. S1P and its close analog sphinganine-1-phosphate (dihydro-S1P) bind to S1PRs, a family of five G-protein-coupled receptors that includes S1P1/EDG-1, S1P2/EDG-5, S1P3/EDG-3, S1P4/EDG-6, and S1P5/EDG-8 (2)
. These S1PRs are coupled to all known G-proteins, thereby endowing extracellular S1P with the ability to regulate a large variety of cellular functions (3
4
5
6)
. Because all these S1PRs except S1P5 are expressed in smooth muscle cells, we have examined whether S1P can modulate HASM cell functions that are critically important in the pathobiology of asthma (1)
. S1P induced airway smooth muscle cell proliferation and augmented EGF-induced DNA proliferation (7)
. By modulating adenylyl cyclase activity and cAMP accumulation, S1P also had potent effects on cytokine secretion. Although S1P inhibited TNF-
-induced RANTES release, it induced substantial IL-6 secretion alone and augmented production of IL-6 induced by TNF- (1)
.
Remodeling of the airway with structural alterations in the bronchial wall such that it becomes hypersensitive to contractile stimuli is a characteristic feature of chronic asthma. Because it is widely accepted that ASM contraction plays a key role in asthmatic attacks, it was of interest to determine whether S1P also regulates HASM cell contractility. Using both 3-dimensional collagen matrices embedded with HASM cells and direct micrometric measurements of cell length, we show that S1P can directly stimulate HASM cell contraction. S1P stimulated contraction independent of ERK1/2 and protein kinase C (PKC), signaling pathways that have been implicated as important components of ASM contraction (8
, 9)
. However, S1P-induced calcium influx from extracellular sources was essential for its effect. S1P might not only regulate HASM hyperplasia and cytokine secretion (1)
, but also contribute to the etiology of asthma by its effects on ASM contractility.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HASM cell culture
Human trachea were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings at the University of Pennsylvania. HASM cells were isolated, purified, and cultured in Ham’s F12 media supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Biofluids) as described previously (1)
. Confluent HASM cells were growth-arrested by incubating monolayers or collagen matrices in Ham’s F12 with 0.1% BSA for 48 h.
Collagen matrix contraction
Confluent HASM cells were released from culture flasks with 0.25% trypsin supplemented with 1 mM EDTA and trypsin was neutralized with F12 media containing 10% FBS. Cells were washed once in serum-free media, counted, and resuspended at a concentration of 1.5 x 106 cells/mL in DMEM. Cells were diluted in neutralized collagen solutions [1.6 mg/mL, prepared using Vitrogen 100 as described previously (10)
] to a concentration of 3 x 105 cells/mL and warmed at 37°C for 3-4 min. Aliquots (400 µL) were distributed in 12-well plates with 19 mm diameter circular scores. After 1 h of polymerization at 37°C, resulting collagen matrices had an area of 
280 mm2. Freshly prepared collagen matrices were then overlaid with F12 media containing 10% FBS and incubated for 2 days to allow mechanical load to develop. HASM cells embedded in collagen matrices were then starved overnight in serum-free F12 media. All treatments for contraction experiments were in DMEM. In some experiments, collagen matrices were pretreated for 2 h with pertussis toxin or for 30 min with all other inhibitors before the initiation of contraction.
Collagen matrices were released from the culture dish, removing isometric load, to initiate contraction. Matrix contraction was allowed to proceed for 2 h at 37°C in the presence or absence of agonists and stopped by fixation in 3.7% formaldehyde/PBS. Collagen matrix areas were subsequently calculated from scanner-generated digital images using NIH Image. Contraction is expressed as the change in mean area relative to unstimulated cells.
Measurement of muscle cell contraction by scanning micrometry
Smooth muscle contraction was measured by scanning micrometry as described previously (11)
. HASM cells were sparsely plated onto glass coverslips at 2 x 102 cells/coverslip and cultured overnight. The cells were serum-starved for 16–20 h in serum-free Hams’ F-12 medium. Cells were then stimulated for 5 min with various agonists. After fixation with 1% acrolein, the mean length of muscle cells treated with S1P or carbachol was compared with the mean length of untreated cells and contraction was expressed as a percentage of decrease in mean cell length.
Myosin light chain phosphorylation
Myosin light chain phosphorylation was assessed by mobility shift in urea/glycerol-PAGE gels (12)
. HASM were cultured in 100 mm culture dishes and cells scraped in 10% (w/v) trichloroacetic acid containing 10 mM dithiothreitol. Samples were incubated overnight at 4°C, then centrifuged for 15 min at 12,000 x g. Pellets were extracted three times with diethylether and resuspended in urea sample buffer (10 mM dithiothreitol, 0.004% bromphenol blue, 8 M Urea, 20 mM Tris, 23 mM glycine, pH 8.6). Proteins were separated on 10% polyacrylamide/40% glycerol gels at 220 V for 16 h at 4°C, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-myosin light chain monoclonal MY-21 antibody (Sigma).
ERK activation
Matrices were placed in 100 µL of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 2 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 5 µg/mL leupeptin, 5 µg/mL aprotinin), homogenized with a Dounce homogenizer (Wheaton Scientific, Millville, NJ, USA), and centrifuged at 14,000 x g for 5 min. Equal amounts of supernatant proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, then immunoblotted with anti-phospho-ERK antibody (1:1000), stripped, and reprobed with anti-total ERK1 antibody (1:5000).
Rho activation
Cytosolic extracts in buffer containing 25 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and 10 µg/mL each aprotinin and leupeptin were incubated at 4°C for 30 min with freshly prepared GST-Rhotekin fusion protein bound to glutathione-agarose beads (a gift from J. S. Gutkind). Bound proteins were washed twice, separated on 15% SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Rho antibody (Santa Cruz). Immunocomplexes were visualized by ECL. GTP-Rho was quantified using NIH Image software and normalized with total cellular Rho.
Fluorescence microscopy
HASM cells were plated on glass coverslips in 12-well dishes (2x104 cells/well), incubated overnight in HAM’s F12 media containing 10% FBS, and subsequently starved for 2 days in serum-free HAM’s F12 media. After treatments, cells were fixed in 3.7% formaldehyde for 10 min at room temperature and permeabilized in 0.5% Triton X-100 for 5 min. Actin filaments were visualized with Alexa 488-conjugated phalloidin (Molecular Probes, Eugene, OR, USA). After washing three times with PBS, coverslips were mounted on slides using an Anti-Fade kit (Molecular Probes) and examined with a confocal laser scanning microscope (Olympus Fluoview).
Cytosolic free calcium
Near-confluent, growth-arrested HASM cells were loaded with 3 µM Fura-2AM as described (13)
and resuspended (at 106cells/mL) in 1 cm quartz cuvettes. After preincubating at 37°C for 2 min with gentle stirring, changes in Fura fluorescence were measured with a fluorescence spectrophotometer (SLM/Aminco, Model AB2, Rochester, NY, USA) after addition of S1P, thrombin, or bradykinin. Thrombin and bradykinin were used as positive controls as these agonists have previously shown to increase calcium mobilization in HASM cells (13)
.
Reproducibility of data
Experiments were repeated at least three times with consistent results and statistical differences were determined by ANOVA.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, it was important to determine whether S1P influences HASM contraction. Adapting a 3-dimensional collagen matrix cell culture system (10)
, we embedded HASM cells in substratum-attached collagen matrices. Like other contractile cells, HASM cells embedded in attached collagen matrices generate mechanical loading forces (isometric tension). Release from the culture dish causes mechanical unloading and contraction as the actin cytoskeleton of the embedded cells becomes free to contract the deformable collagen lattice (10)
.
Although matrix contraction in floating collagen gel systems by fibroblasts (10)
and smooth muscle cells (14)
is a function of collagen remodeling, smooth muscle cell contraction of collagen matrices after the development of mechanical load is in large part similar to actomyosin-driven smooth muscle contraction (15)
. Smooth muscle cells cultured under conditions of isometric load become spindle-shaped, contain stress fibers, and are aligned along lines of stress. Upon release of this mechanical load, stress fibers become free to contract, allowing cells to markedly reduce the size of the collagen lattices in which they are embedded (16)
.
HASM cells contracted the collagen lattices from an original area of 240 mm2 to an area of 120 mm2 within 2 h after release in serum-free media. This basal contraction was enhanced by known stimulators of HASM contractility such as bradykinin and thrombin (Fig. 1
A). S1P induced contraction of collagen matrices to a similar extent as bradykinin, but was somewhat less effective than thrombin or serum (Fig. 1A
). Moreover, S1P-mediated contraction was detectable at a concentration as low as 1 nM, and began to plateau after 10 nM (Fig. 1B
). Dihydro-S1P, which is identical to S1P and only lacks the 4,5-trans double bond, yet binds and activates S1PRs with lower affinity than S1P (17)
, also stimulated collagen matrix contraction. However, dihydro-S1P was less potent than S1P and, at 10 nM, did not induce significant contraction; at concentrations >100 nM, it was as effective as S1P (Fig. 1B
).
|
Cells in stressed matrices exhibit a spread morphology with prominent stress fibers and retract during contraction as the stress fibers transition to an actin meshwork (10)
. The actin fibers themselves can contract once there is no longer a rigid substratum to maintain isometric tension (10)
. Similar to other cell types (18)
, including arterial smooth muscle cells (19)
, S1P potently induced stress fiber formation in HASM cells (Fig. 2
).
|
Role of Gi and ERK1/2 in S1P-stimulated contraction
Nanomolar concentrations of S1P regulate cell functions through G-protein-coupled receptors (GPCRs) of the S1PR family (4
5
6)
. Besides providing direct stimulation of contraction, Gi-coupled muscarinic receptors prevent adenylyl cyclase stimulation and increases in cAMP that might block airway smooth muscle cell contraction (20
, 21)
. Surprisingly, although HASM cells express S1P1 and S1P4 (1)
, which are coupled mainly to Gi (22)
, pretreatment of HASM cells with pertussis toxin, which ADP ribosylates and inactivates Gi, did not inhibit S1P-mediated collagen matrix contraction (Fig. 3
A). However, pertussis toxin effectively blocked S1P-stimulated ERK1/2 activation as determined with phosphospecific antibody (Fig. 3B
), in agreement with previous studies showing that this is a Gi-coupled S1PR pathway (22)
. Because activation of ERK plays an important role in smooth muscle cell contraction induced by various agonists (23
, 24)
, we also examined the effect of inhibition of ERK1/2. Although it had no effect on basal contraction, PD98059, a MEK1/2 inhibitor, enhanced rather than inhibited S1P-mediated contraction (Fig. 3C
). Yet, as expected, this inhibitor blocked S1P-induced ERK1/2 activation (data not shown). In contrast and in agreement with a previous study (25)
, HASM contraction induced by carbachol at a concentration that elicited maximum contraction (data not shown) was reduced by PD98059. Thus, not only is S1P-stimulated contractility of HASM cells independent of the Gi pathway, the ERK pathway is dispensable for this effect.
|
Calcium mobilization and HASM contraction
Because calcium mobilization is a major regulator of ASM contractility (13)
, we determined whether calcium is required for HASM cell contraction of collagen matrices. Removal of calcium from the medium with EGTA completely blocked S1P-mediated collagen matrix contraction (Fig. 4
A). When calcium was added back, the ability of S1P to induce collagen matrix contraction was restored. Similar results were obtained with calcium-free media where S1P-induced contractions were increased in a calcium concentration-dependent manner (Fig. 4B
). Calcium influx mediated by the calcium ionophore A23187 also caused an increase in collagen matrix contraction (Fig. 4C
).
|
As calcium is required for S1P-mediated HASM cell collagen matrix contraction, we next determined whether nanomolar concentrations of S1P can regulate calcium levels in HASM cells. Indeed, S1P induced calcium mobilization in HASM cells at a concentration as low as 10 nM and this effect was markedly increased at higher concentrations (Fig. 4D
). Repeated stimulation with S1P did not produce attenuation of the calcium signal (Fig. 4E
), in contrast to other cell types (3
, 26)
. Moreover, similar to our previous results with HASM cells (1)
, pretreatment with pertussis toxin only slightly inhibited S1P-induced calcium elevation. Changes in [Ca2+]i induced by 200 nM S1P in the absence or presence of pretreatment with 200 ng/mL pertussis toxin for 2 h were 220 ± 13 and 165 ± 11 nM, respectively.
Role of calcium influx in S1P-mediated HASM cell contractility
Collectively, our results suggest that calcium is critical for S1P-induced HASM cell contraction. Surprisingly, pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores did not completely block increases in [Ca2+]i induced by S1P (Fig. 5
A), but completely blocked bradykinin-induced calcium mobilization (data not shown), suggesting the involvement of thapsigargin-insensitive calcium stores. Moreover, short-term removal of extracellular calcium without depleting intracellular stores only partially reduced S1P-induced [Ca2+]i (Fig. 5A
). These results suggest that S1P might increase [Ca2+]i by stimulating calcium influx.
|
Next we examined whether calcium influx and/or mobilization are important for S1P-induced HASM cell contraction. Collagen matrices were preincubated with EGTA for 20 min before initiation of contraction to reduce both extracellular and intracellular calcium. This inhibited S1P-induced HASM contractions to a much greater extent compared with the effect of adding EGTA simultaneously with S1P, which only removes extracellular calcium (Fig. 5B
). To further determine whether intracellular calcium stores may be important in HASM contraction, collagen matrices were pretreated with thapsigargin before S1P stimulation. Thapsigargin did not inhibit S1P-stimulated contraction while blocking carbachol-induced responses (Fig. 5C
). We used the calcium channel blocker verapamil to investigate the possibility that extracellular calcium influx is required for HASM contraction. Indeed, verapamil markedly reduced S1P-induced contraction whereas contraction induced by carbachol was not influenced by this calcium channel inhibitor (Fig. 5D
).
Role of Rho in S1P-induced HASM contractility
GPCRs activated by a wide variety of agonists can switch on myosin light chain phosphorylation and force in smooth muscle. The extent of myosin light chain phosphorylation reflects activity of the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and the myosin light chain phosphatase (27
, 28)
. Thus, it was important to examine the relationship between MLC20 phosphorylation and S1P-induced contraction. Basal levels of phosphorylated MLC20 were very low in HASM cells. Treatment with S1P for as little as 2 min resulted in marked MLC20 phosphorylation, with increased levels of both the mono- and diphosphorylated forms and concomitant disappearance of the unphosphorylated MLC (Fig. 6
A).
|
At constant calcium and MLCK activity, signals that inhibit myosin phosphatase activity cause a leftward shift of the calcium force-response curve, a physiological relevant phenomenon known as calcium sensitization. Both the small GTPase Rho, which stimulates Rho-associated kinase (ROK), and protein kinase C (PKC) pathways converge on inhibition of MLC phosphatase and thus increase levels of phosphorylated MLC (9
, 28)
. Because both S1P2 and S1P3 can activate the Rho pathway and stress fiber formation in a variety of cell types, including vascular smooth muscle cells (29)
, we examined the involvement of this pathway. The ROK inhibitor Y-27632 nearly completely prevented phosphorylation of MLC20 induced by S1P (Fig. 6A
). Next we examined the cellular amount of the GTP-bound, active form of Rho as measured by a specific GTP-Rho pulldown assay. Carbachol markedly and rapidly stimulated Rho, which was still evident after 10 min. S1P increased GTP-Rho only transiently, with a maximum effect between 3 and 5 min, and to a much lesser extent than carbachol (Fig. 6B
).
Rho kinase and PKC in S1P-iInduced contraction of individual HASM cells
HASM-populated collagen matrices undergo contraction within 2 h upon release from isometric load, suggesting this mainly involves actin–myosin contractility mechanisms rather than matrix remodeling. Nonetheless, to examine contraction in more detail, we measured changes in smooth muscle cell length by a micrometric method that permits rapid continuous contractility measurements in single smooth muscle cells and has been used extensively to examine contraction of many types of isolated smooth muscle cells (11)
. S1P caused a rapid contraction of HASM cells that was evident within 5 min, reaching a maximum by 20 min (Fig. 7
A). The effect of S1P was concentration dependent and a maximal response was observed at 100 nM (Fig. 7B
). The sensitivity to S1P was similar to the collagen matrix contraction assay (Fig. 1B
), although the concentration-contraction curve was shifted to the right. Moreover, contraction induced by S1P at 5 min in HASM cells was abolished by incubation in calcium-free medium containing EGTA (Fig. 7B
), in agreement with a dependence on extracellular calcium for S1P-mediated HASM cell collagen matrix contraction (Fig. 4A, B
). The ROK inhibitor Y-27632 did not change cell length by itself. Although Y-27632 had no effect on S1P-induced contraction at 5 min (Fig. 7B
), it significantly inhibited the effect of S1P at 20 min (Fig. 7C
). Even the sustained contraction induced by S1P at 30 min was markedly inhibited by Y-2763 (Fig. 7C
).
|
There is abundant evidence that PKC mediates agonist-induced increases in smooth muscle calcium sensitivity (28)
. Similar to carbachol and S1P, acute activation of PKC with the phorbol ester TPA stimulated HASM contraction (Fig. 8
). To examine the role of PKC, we used bisindolylmaleimide I, a highly selective, cell-permeable PKC inhibitor that is a competitive inhibitor of the ATP binding site and calphostin C, which targets the regulatory subunit of both classical and novel forms of PKC (30)
. As expected, bisindolylmaleimide I and calphostin C blocked TPA stimulated HASM contraction. Neither bisindolylmaleimide I nor calphostin C blocked S1P-stimulated contraction (Fig. 8)
. By contrast, both PKC inhibitors prevented carbachol-induced contraction.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. These increased S1P concentrations are near the Kd values of S1PRs for S1P and are sufficient to significantly stimulate contraction of HASM cells. Contraction of HASM was observed at much lower S1P concentrations than those required for its effects on DNA synthesis and proliferation, inhibition of TNF--induced RANTES release, and IL-6 secretion in these cells, all of which required a 10 micromolar concentration (1)
. However, this might be due to addition of S1P in DMSO rather than as a complex with BSA as used in the current study.
S1P induced increases in [Ca2+]i in vascular smooth muscle and caused vasoconstriction of isolated rat microvessels via receptors coupled to pertussis toxin-sensitive G-proteins (31)
. We found that S1P also increased [Ca2+]i and activated contraction of HASM cells in a calcium-dependent manner. Although S1P mobilized calcium to a much lesser extent than bradykinin or thrombin (1)
, it was nearly as effective in provoking HASM contraction. In vascular smooth muscle myocytes from cerebral arteries, S1P induces a global increase in [Ca2+]i, probably as the result of release from intracellular thapsigargin-sensitive stores, because it is does not occur after inhibition of the sarcoplasmic reticulum Ca2+-ATPase (32)
. Moreover, removal of external calcium or inhibition of voltage-gated calcium channels had no significant effect on the S1P-induced calcium response (32)
. In HASM cells, however, we found that depletion of intracellular calcium stores with thapsigargin only partially reduced S1P-induced [Ca2+]i and had no effect on their contractility. In contrast, S1P-induced HASM cell contractility is markedly decreased in calcium-free medium or by verapamil, an L-type calcium channel blocker, strongly suggesting that S1P-mediated contraction involves influx of calcium from extracellular sources. In agreement, previous studies in thyroid FRTL-5 cells (33)
and myoblasts (34)
have shown that the rise in calcium was strictly dependent on its availability from extracellular sources. However, S1P induced contraction of C2C12 myoblastic cells by calcium-independent mechanisms (35)
. Whereas previous reports demonstrated desensitization of calcium responses upon repeated additions of S1P in many types of cells (3
, 26)
, we found that calcium signals in HASM cells were not attenuated by repeated pulses of S1P. This could have important implications for the regulation of HASM contractile function and bronchial hyper-responsiveness, a defined feature of asthma, in light of the elevated level of S1P in the asthmatic airway after allergen stimulation (1)
.
In contrast to the requirement for calcium in S1P-induced HASM cell collagen matrix contraction, inhibition of PKC or ERK had no significant effects, suggesting that S1P-induced contractility is mediated mainly via calcium calmodulin-dependent kinases, which, upon calcium binding, phosphorylate and activate MLCK (27
, 28)
. In contrast, we found that carbachol-induced contraction was dependent on activation of ERK and PKC. It is well established that ROK phosphorylates and inactivates MLC phosphatase, thereby increasing phosphorylation of MLC20 leading to contraction of the actomyosin-based cytoskeleton and calcium sensitization of smooth muscle cells (27)
. S1P stimulated Rho, albeit to a lesser and more transient extent than carbachol. Using the selective ROK inhibitor Y-27632, we showed that ROK is involved in the late, but not acute, phase of S1P-induced contraction. In agreement with our finding, S1P induces vasoconstriction in the canine basilar artery in vitro and in vivo through a mechanism involving activation of the Rho/ROK pathway (36)
. S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction (32)
. However in aorta, S1P did not produce constriction or RhoA activation (32)
. This differential signaling may be related to the expression of S1P receptor subtypes as the expression of S1P3 and S1P2 is fourfold higher in cerebral artery than in aorta, whereas S1P1 expression is similar in both types of vascular smooth muscle cells. Therefore, the ability of S1P to act as a vasoactive mediator may depend on the receptors expressed by a particular target tissue.
S1PR signaling in S1P-induced contraction
We found that nanomolar concentrations of S1P effectively mediated HASM cell contraction compared with the micromolar concentrations required for intracellular effects (37)
, suggesting the involvement of high affinity S1P receptors. Support for a receptor-mediated process is further bolstered by the fact that dihydro-S1P, a S1P analog with no known intracellular effects (38)
, yet binds and signals through S1PRs (37)
, could also stimulate contraction of HASM cells, which express S1P1–4 (1)
. The observation that pertussis toxin does not block S1P-induced calcium mobilization in HASM cells or contraction of collagen matrices strongly excludes the involvement of S1P1 and S1P4, as these receptors are mainly Gi-coupled (39
, 40)
. Thus, S1P2 or S1P3 are the most likely S1PRs to mediate Rho- and calcium-dependent S1P-stimulated HASM cell contraction. Indeed, studies with fibroblasts from S1P2 and S1P3 knockout mice indicate that S1P2 and S1P3 are preferentially coupled to the Rho and PLC pathways, respectively (41)
. Dihydro-S1P and S1P have similar binding affinities with all of the S1PRs, except S1P2, which has a 20-fold lower affinity for dihydro-S1P (17)
. In view of the lower potency of dihydro-S1P to induce matrix contraction and the minimal InsP3 production induced by S1P in HASM cells (1)
, it is tempting to speculate that S1P2 may mediate S1P-induced HASM cell contractility. Further studies are necessary to confirm the specific functions of these receptors on HASM.
In light of the ability of S1P to stimulate HASM cell growth and proinflammatory cytokine production (1)
, as well as calcium mobilization and contraction, the presence of S1P in the asthmatic airways suggests a potential role for this sphingolipid in orchestrating both the acute asthmatic bronchoconstriction reaction and the chronic features of airway remodeling.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication September 16, 2002. Accepted for publication June 13, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Maceyka, S. E. Alvarez, S. Milstien, and S. Spiegel Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration Mol. Cell. Biol., September 15, 2008; 28(18): 5687 - 5697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hashimoto, X. Wang, L. Mao, T. Kobayashi, S. Kawasaki, N. Mori, M. L. Toews, H. J. Kim, D. R. Cerutis, X. Liu, et al. Sphingosine 1-Phosphate Potentiates Human Lung Fibroblast Chemotaxis through the S1P2 Receptor Am. J. Respir. Cell Mol. Biol., September 1, 2008; 39(3): 356 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Wamhoff, K. R. Lynch, T. L. Macdonald, and G. K. Owens Sphingosine-1-Phosphate Receptor Subtypes Differentially Regulate Smooth Muscle Cell Phenotype Arterioscler. Thromb. Vasc. Biol., August 1, 2008; 28(8): 1454 - 1461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nishiuma, Y. Nishimura, T. Okada, E. Kuramoto, Y. Kotani, S. Jahangeer, and S.-i. Nakamura Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1085 - L1093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-Q. Lai, H. H. Goh, Z. Bao, W. S. F. Wong, A. J. Melendez, and B. P. Leung The Role of Sphingosine Kinase in a Murine Model of Allergic Asthma J. Immunol., March 15, 2008; 180(6): 4323 - 4329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Kassel, N. A. Schulte, S. M. Parker, A. D. Lanik, and M. L. Toews Lysophosphatidic Acid Decreases Epidermal Growth Factor Receptor Binding in Airway Epithelial Cells J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 109 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Matsumoto, L. M Moir, B. G G Oliver, J. K Burgess, M. Roth, J. L Black, and B. E McParland Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma Thorax, October 1, 2007; 62(10): 848 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Watterson, K. M. Berg, D. Kapitonov, S. G. Payne, A. S. Miner, R. Bittman, S. Milstien, P. H. Ratz, and S. Spiegel Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone FASEB J, September 1, 2007; 21(11): 2818 - 2828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Goetzl Diverse pathways for nuclear signaling by G protein-coupled receptors and their ligands FASEB J, March 1, 2007; 21(3): 638 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kume, N. Takeda, T. Oguma, S. Ito, M. Kondo, Y. Ito, and K. Shimokata Sphingosine 1-Phosphate Causes Airway Hyper-Reactivity by Rho-Mediated Myosin Phosphatase Inactivation J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 766 - 773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Leiber, Y. Banno, and Z. Tanfin Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms Am J Physiol Cell Physiol, January 1, 2007; 292(1): C240 - C250. [Abstract] [Full Text] [PDF]
|
|
|
|
