QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.118042v1 321/3/1102    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirakawa, M. Articles by Oike, M. Search for Related Content PubMed PubMed Citation Articles by Hirakawa, M. Articles by Oike, M.

GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Protein Kinase A Inhibits Lysophosphatidic Acid-Induced Migration of Airway Smooth Muscle Cells

Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Received December 1, 2006; accepted March 7, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Lysophosphatidic acid (LPA) is a bioactive phospholipid that is released from activated platelets and affects contractile properties of airway smooth muscle cells. However, possible roles of LPA on cell migration, one of the initial events of airway remodeling, are not clarified. This study aimed to examine the effects of LPA on migration and actin fiber formation in bovine tracheal smooth muscle cells (BTSMCs). Random and oriented cell migrations were examined with wound assay and Boyden chamber assay, respectively. Cytosolic actin fibers were stained with rhodamine-phalloidin. Membrane translocation of RhoA, a hallmark of RhoA activation, was assessed by Western blotting. LPA augmented the migration of BTSMCs from wounded confluent monolayer but did not accelerate the chemotactic migration toward LPA. LPA also induced a transient actin reorganization and RhoA activation. Dense actin fibers were observed mainly in the wound edge but not in migrated cells, thereby suggesting the role of actin reorganization in the initiation of cell migration. LPA-induced actin fiber formation was blocked by Y27632 [R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide], an inhibitor of Rho kinase. Effects of LPA on migration and actin fiber formation were also inhibited by cAMP-elevating agents, i.e., dibutyryl cAMP, forskolin, isoproterenol, and theophylline. KT5720 (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], a protein kinase A (PKA) inhibitor, reversed the inhibitory actins of cAMP on LPA-induced responses. These results indicate that LPA induces cAMP/PKA-sensitive, RhoA-mediated random migration of BTSMCs. Regulation of this mechanism would be beneficial for the control of airway remodeling.

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that is generated in serum (Xie et al., 2002) or secreted from platelets (Moolenaar, 1995) and serves as an autocrine and paracrine mediator. Because increased vascular permeability with plasma leakage is a feature of asthma (Wilson, 2000), the concentration of LPA is supposed to be increased in injured and/or asthmatic airway. Potential roles of LPA in the development of respiratory diseases, especially asthma, have been suggested (Toews et al., 2002). LPA binds to its specific receptors and activates small GTPase family protein RhoA and subsequent Rho kinase (Moolenaar, 1995). It is generally accepted that cell migration requires cytoskeletal remodeling, for which small GTPase family proteins play an essential role (Ridley, 2001). Therefore, it can be hypothesized that LPA may induce airway smooth muscle migration, thereby initiating airway remodeling. Actually, LPA is reported to induce structural remodeling (Cerutis et al., 1997) as well as Ca²⁺ hypersensitivity (Toews et al., 1997; Sakai et al., 2003) in airway smooth muscle cells in vitro.

The aim of this study was to investigate the effects of LPA on cell migration and actin reorganization in bovine tracheal smooth muscle cells (BTSMCs). Our results revealed, for the first time, that LPA induces random migration of airway smooth muscle cells via RhoA-mediated, protein kinase A (PKA)-sensitive actin reorganization.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture. Tracheas of 1-year-old calves were obtained from a local slaughterhouse. Smooth muscle cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum by explant method (Sakai et al., 2003). Cells grown in confluence were harvested by trypsin digestion and stored at -80°C after one-step subculture. Present study was performed with BTSMCs obtained from seven tracheas. We have confirmed with Western blotting that the BTSMCs used in the present study expressed smooth muscle marker proteins, i.e., calponin, myosin, and -smooth muscle actin (data not shown).

Cell Migration Assays. Migration of BTSMCs was assessed with a wound assay (Kimura et al., 2001) and a Boyden chamber assay (John et al., 1997). In the wound assay, cells grown in confluent on coverslips were scraped with an edge of fine razor. The wound edge was viewed and photographed under a microscope (Eclipse TS100; Nikon, Tokyo, Japan) before and after culturing for 24 h in serum-free DMEM in the presence of 10 µM mitomycin C. The number of the cells migrated from the wound surface was then manually counted. In some experiments, actin cytoskeleton was stained in the wound edge as described below.

In the Boyden chamber assay, 10⁵ cells were seeded on 6.5-mm diameter Transwell filter cups (8-µm pore; Corning Life Sciences, Acton, MA) with 100 µl of serum-free DMEM and incubated for 6 h at 37°C in the presence of 1 µM LPA either in the filter cup (i.e., upper side of the filter), in the well (i.e., lower side of the filter), or in both chambers. Control wells were incubated in the absence of LPA. The filter was removed from the cup, fixed with 70% ethanol, and stained with hematoxylin. The number of cells migrated to the lower side of the filter was manually counted under microscope.

Staining of Actin Cytoskeleton. Reorganization of actin cytoskeleton was assessed with F-actin staining (Koyama et al., 2001). Cells were fixed with 2% paraformaldehyde after each treatment and permeabilized with 0.1% Triton X-100. Rhodamine-conjugated phalloidin (Invitrogen, Carlsbad, CA) was then applied for 20 min after blocking nonspecific binding with 0.5% fetal bovine serum. F-actin fibers were then observed with fluorescence microscope (Eclipse E600; Nikon).

Measurement of Intracellular cAMP Concentration. Intracellular cAMP concentration ([cAMP]_i) was measured with enzyme immunoassay by using a commercial kit (Biotrak cAMP EIA system; GE Healthcare, Little Chalfont, Buckinghamshire, UK). BTSMCs were cultured overnight at a density of 5000 cells/well in 96-well culture plates, and [cAMP]_i was measured according to the manufacturer's instructions.

Western Blot Analysis of RhoA Activation. Activation of RhoA was assessed with enhanced chemiluminescence Western blotting. BTSMCs were pretreated with theophylline, dibutyryl cAMP, forskolin, or vehicle for 1 h. Cells were then lysed with 1% Triton X-100 immediately or after applying 1 µM LPA for 5 min. Cell lysate was centrifuged at 100,000g for 1 h, and the pellet was harvested as a membrane fraction. A constant amount of membrane fraction (50 µg protein/lane) was separated with SDS-polyacrylamide gel electrophoresis, and enhanced chemiluminescence Western blotting was performed by using anti-RhoA antibody (Cytoskeleton, Inc., Denver, CO) and the antibody against -actin (Sigma, St. Louis, MO) that was measured as an internal control. Band images were analyzed with a lumino image analyzer (FAS-1000; Toyobo, Osaka, Japan).

Drugs and Solution. The modified Krebs' solution used in the present experiment was 132.4 mM NaCl, 5.9 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 11.5 mM glucose, and 11.5 mM HEPES, and pH was adjusted to 7.4 with NaOH. LPA (Sigma) was dissolved at 3 mM in chloroform/methanol. Solvent was evaporated by puffing nitrogen gas and sonicated after adding Krebs' solution or DMEM to make 20 µM LPA solution. Then, the solution was further diluted to 1 µM shortly before use. ATP, theophylline, forskolin, dibutyryl cAMP, Y27632, and KT5720 were purchased from Sigma.

Statistics. Data were expressed with mean ± S.E.M. values. Statistical significance was examined with Student's unpaired t test. Probability below 0.05 (p < 0.05) was considered as significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
LPA-Induced Migration from the Wound Edge in BTSMCs. Firstly, we examined the effects of LPA on the random migration of BTSMCs with the wound assay. LPA (1 µM) induced a marked acceleration of cell migration into the cell-free area (Fig. 1A). The number of the migrated cells after 24 h was significantly increased by LPA (control, 33 ± 3 cells/0.5-mm wound surface, n = 6; 1 µM LPA, 79 ± 8 cells/0.5-mm wound surface, n = 6, p < 0.01 versus control, see also Fig. 4A).

View larger version (92K): [in this window] [in a new window]   Fig. 1. Migration of BTSMCs induced by LPA. A, cell migration from wound edge after culturing for 24 h in the presence and absence of 1 µM LPA. Dotted lines, wound edge. Scales, 200 µm. B, oriented migration of BTSMCs was examined with Boyden chamber assay. BTSMCs (105 cells) were put in the filter cup and incubated for 6 h in the presence or absence of 1 µM LPA. LPA was applied in the filter cup, in the well, or in both chambers. The cells that migrated to the lower surface of the filter were observed after staining with hematoxylin. Representative images obtained in the absence and presence of LPA in the filter cup are shown. Note that the fibroblast-like adherent cells are the migrated cells. Scales, 50 µm. Bar graph, statistical analysis of the number of migrated cells. *, p < 0.05; **, p < 0.01; n.s., p > 0.05 versus control. Data were obtained from six repeated experiments for each condition.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Statistical analysis of LPA-induced migration of BTSMCs. A, confluent BTSMCs monolayer was wounded and cultured in the presence or absence of 1 µM LPA, and the effects of Y27632 (10 µM), dibutyryl cAMP (300 µM), forskolin (30 µM), theophylline (10 µM), or isoproterenol (10 µM) were examined. The number of cells migrated to the cell-free area was counted after 24 h (n = 6 for each condition). **, p < 0.01 versus control. , p < 0.01 versus LPA alone. B, effects of KT5720 on the inhibition of LPA-induced migration by cAMP-elevating agents and Y27632 were examined. LPA (1 µM) was applied to the wounded monolayer in the presence of 0.1, 1, or 10 µM KT5720, and the number of migrated cells was counted after 24 h. Data were obtained from six repeated measurements.

Reorganization of Actin Cytoskeleton by LPA in BTSMCs. The above results suggest that LPA activates random migration of BTSMCs. Because cell migration is obtained by the reorganization of actin cytoskeleton, we then examined the effects of LPA on actin fiber formation in BTSMCs. LPA (1 µM) induced a formation of dense actin fibers within 5 min in subconfluent BTSMCs. Actin fibers were persisted at least for 10 min and converged into peripheral ruffles in 30 min (Fig. 2A). Thickness of actin fibers depends on the concentration of LPA (Fig. 2A), suggesting that the actin fiber formation was not due to mechanical stress artificially generated by solution exchange.

View larger version (107K): [in this window] [in a new window]   Fig. 2. Effects of LPA on actin cytoskeleton in BTSMCs. A, LPA (0.1, 1, or 10 µM) was applied to nonconfluent control BTSMCs for 5, 10, or 30 min, and actin fibers were stained with rhodamine-phalloidin. Representative images from 4 to 12 repeated experiments are shown for each condition. Scales, 20 µm. B, actin fibers were stained in the wound assay. Note that dense actin fibers were observed in the wound surface but not in confluent monolayer or migrated cells. Scale, 100 µm. C, higher magnification of actin fibers of the migrated cells and the wound surface. Arrow, direction of migration. Scale, 20 µm.

Effects of Rho Kinase Inhibitor on LPA-Induced Responses in BTSMCs. LPA is known to activate small G-protein RhoA and subsequent Rho kinase (Moolenaar, 1995), so we then examined the effects of Rho kinase inhibitor Y27632 on LPA-induced migration and actin reorganization. In the wound assay, the LPA-induced increase in migration was significantly reversed by 10 µM Y27632 (37 ± 3 cells/0.5-mm wound surface, p < 0.01 versus LPA alone, n = 6, see also Fig. 4A). LPA-induced actin reorganization was also inhibited by the treatment with 10 µM Y27632 for 1 h (Fig. 3A). These suggest the involvement of RhoA/Rho kinase pathway in LPA-induced migration and actin formation.

View larger version (100K): [in this window] [in a new window]   Fig. 3. Effects of Rho kinase inhibitor, cAMP, and protein kinase A inhibitor on LPA-induced actin fiber formation in BTSMCs. A, cells were treated with 10 µM Y27632, and actin fibers were stained before or after the application of 1 µM LPA for 5 min. B, LPA (1 µM) was applied for 5 min to the cells treated with dibutyryl cAMP (300 µM), forskolin (30 µM), theophylline (10 µM), or isoproterenol (10 µM) for 1 h. LPA-induced actin formation in untreated cells is also demonstrated. C, BTSMCs were pretreated with 1 µM KT5720 for 1 h in the absence or presence of dibutyryl cAMP (300 µM), forskolin (30 µM), theophylline (10 µM), or isoproterenol (10 µM), and the LPA (1 µM)-induced actin fiber formations were examined. Representative images from four to six repeated experiments are shown for each condition. Scales, 20 µm.

LPA-induced increase in the migration of BTSMCs was significantly suppressed by 300 µM dibutyryl cAMP, 30 µM forskolin, 10 µM theophylline, and 10 µM isoproterenol (Fig. 4A). Furthermore, these agents also abolished LPA-induced actin reorganization (Fig. 3B). These agents, however, did not affect the migration (Fig. 4A) and actin cytoskeleton (not shown) of control cells.

Measurement of the Intracellular cAMP Concentration in BTSMCs. Next, we measured [cAMP]_i in the presence or absence of dibutyryl cAMP, forskolin, and theophylline. [cAMP]_i was 94 ± 14 fmol/well in control BTSMCs and was elevated to 3100 ± 310 and 700 ± 130 fmol/well by forskolin (30 µM) and theophylline (10 µM), respectively (n = 6 for each condition). Membrane-permeated dibutyryl cAMP was also detected as "cAMP" by the antibody in the kit and showed 4500 ± 500 fmol/well after the treatment with dibutyryl cAMP (300 µM, n = 6).

Effects of KT5720 on LPA-Induced Migration and Actin Reorganization. Because these suggest that cAMP inhibits LPA-induced migration and actin reorganization in BTSMCs, we examined whether this was obtained directly with cAMP or via PKA. Pretreatment with a PKA inhibitor, KT5720 (1 µM), alone for 1 h did not affect LPA-induced actin fiber formation in BTSMCs (Fig. 3C). The inhibitory effects of Y27632 on LPA-induced actin reorganization were also not affected by KT5720 (Fig. 3C). In contrast, dibutyryl cAMP, forskolin, theophylline, and isoproterenol did not suppress the LPA-induced actin fiber formation in KT5720-treated BTSMCs (Fig. 3C), indicating the PKA-mediated rather than direct effects of cAMP on LPA-induced actin reorganization.

KT5720 also reversed the inhibitory effects of dibutyryl cAMP, forskolin, theophylline, and isoproterenol on LPA-induced migration of BTSMCs from the wound surface (Fig. 4B). Effects of KT5720 were concentration-dependent, and 10 µM KT5720 almost completely reversed the effects of cAMP-elevating agents on LPA-induced migration of BTSMCs. In contrast, 1 µM KT5720 did not reverse the effects of Y27632, indicating that a nonspecific augmentation of cell migration by KT5720 is negligible (Fig. 4B).

LPA-Induced Membrane Translocation of RhoA in BTSMCs. We finally examined the effects of cAMP-elevating agents and KT5720 on LPA-induced RhoA membrane translocation, which is a reflection of RhoA activation. LPA-induced membrane translocation of RhoA was inhibited by dibutyryl cAMP (Fig. 5A) and theophylline (Fig. 5B). KT5720 did not affect either the resting level of RhoA in the membrane fraction or LPA-induced RhoA membrane translocation. On the other hand, KT5720 reversed the inhibitory effects of dibutyryl cAMP (300 µM) and theophylline (10 µM) on LPA-induced membrane translocation of RhoA (Fig. 5).

View larger version (43K): [in this window] [in a new window]   Fig. 5. LPA-induced membrane translocation of RhoA in BTSMCs. BTSMCs were pretreated with or without dibutyryl cAMP (300 µM, A) and theophylline (10 µM, B) for 1 h in the presence or absence of KT5720 (1 µM). Control or LPA (1 µM, 5 min)-treated BTSMCs were lysed, and the membrane fraction was obtained by centrifugation at 100,000g for 1 h. Expressions of RhoA and -actin in the membrane fraction were then examined with Western blotting, and the representative band images are shown in the upper panels. Densitometric analysis of the bands is shown in the lower panels. Each bar represents mean ± S.E.M. value obtained from four repeated experiments. *, p < 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion References

We have also shown that LPA increases the actin fiber formation in nonconfluent BTSMCs (Fig. 2A). Actin cytoskeleton was markedly augmented in the wound edge, whereas cytosolic actin fibers were not prominent in the migrated cells (Fig. 2, B and C). Therefore, although detailed relationship between actin formation and cell migration has yet to be elucidated, this suggests that the formation of dense actin fibers is essential for the initiation of random cell migration in BTSMCs. Y27632, an inhibitor of Rho kinase, inhibited LPA-induced migration (Fig. 4A) and actin reorganization (Fig. 3A). Furthermore, LPA induced a membrane translocation of RhoA (Fig. 5), which is a hallmark of RhoA activation (Kranenburg et al., 1997). A previous report also showed that Y27632 inhibited the platelet-derived growth factor-induced migration of cultured human airway smooth muscle cells (Parameswaran et al., 2002). It is well established that the activation of RhoA/Rho kinase cascade induces actin reorganization (Kaibuchi et al., 1999); thus, we hypothesize that RhoA activation and subsequent actin reorganization play a central role in the LPA-induced migration of BTSMCs. Activation of peroxisome proliferator-activated receptor , another intracellular signal evoked by LPA (McIntyre et al., 2003), is probably not involved in the LPA-induced actin reorganization because peroxisome proliferator-activated receptor activator troglitazone could not mimic the effects of LPA on actin fibers (M. Watanabe and M. Oike, unpublished data).

Goncharova et al. (2003) reported that the migration of human tracheal smooth muscle cells induced by various growth factors is inhibited by cAMP. They speculated that cAMP-responsive element promoter activity was involved in the inhibition of migration (Goncharova et al., 2003). Possible involvement of cAMP-responsive element binding proteins was also suggested in the migration of vascular smooth muscle cells (Klemm et al., 2001). The authors, therefore, speculated that the inhibition of cell migration with cAMP was determined at the level of gene transcription regulation, and the relationship between cAMP-responsive element binding and RhoA-mediated actin cytoskeleton was not considered in their reports. In contrast, another group reported in hepatoma cells that cAMP suppressed the migration due to the inhibition of RhoA (Mukai et al., 2000). In the present study, dibutyryl cAMP, forskolin, and theophylline, all of which were confirmed to induce [cAMP]_i elevation, as well as isoproterenol inhibited LPA-induced migration (Fig. 4A) and actin fiber formation (Fig. 3B) in BTSMCs. Furthermore, dibutyryl cAMP and theophylline inhibited the LPA-induced RhoA activation (Fig. 5). Therefore, we have concluded that the elevation of [cAMP]_i suppresses LPA-induced actin reorganization and subsequent cell migration by inhibiting RhoA activation. KT5720 reversed the inhibitory effects of cAMP on LPA-induced actin reorganization (Fig. 3C), migration (Fig. 4B), and RhoA activation (Fig. 5). However, KT5720 but did not affect the Y27632-induced inhibition of migration (Fig. 4B) and actin reorganization (Fig. 3C). Therefore, the changes in RhoA and actin shown here seem to be a more likely explanation for the effects on cell migration than the transcriptional mechanisms proposed previously.

Previous studies have suggested at least two possibilities for the mechanism of action of PKA on RhoA, i.e., direct phosphorylation of RhoA and the alteration of Dbl proteins. It was reported that PKA specifically phosphorylated RhoA at Ser188 (Lang et al., 1996; Ellerbroek et al., 2003), which would lead to the dissociation of RhoA from the membrane toward the cytosol (Lang et al., 1996). Another possibility is the alteration of Dbl family proteins, which convert inactive RhoA-GDP into active RhoA-GTP (Whitehead et al., 1997). AKAP-Lbc, a Dbl family protein in cardiomyocytes, has an anchoring site for PKA and was activated by LPA (Diviani et al., 2001) and inactivated by the anchored PKA (Diviani et al., 2006). Although it is not known yet whether such PKA-anchoring Dbl proteins are expressed in airway smooth muscle cells or not, PKA may negatively control the functions of Dbl proteins in BTSMCs.

Involvement of LPA in the pathogenesis of airway diseases has recently been considered from various aspects, including smooth muscle mitosis (Cerutis et al., 1997), contractility (Toews et al., 1997), and Ca²⁺ sensitization (Sakai et al., 2003). The present study has revealed, for the first time, that LPA also induces smooth muscle migration that would lead to airway remodeling. Furthermore, this study provides a novel possible mechanism of action of theophylline and probably other cAMP-elevating agents on airway diseases, i.e., the inhibition of smooth muscle migration.

	Footnotes

 
This work was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.118042.

ABBREVIATIONS: LPA, lysophosphatidic acid; BTSMC, bovine tracheal smooth muscle cell; PKA, protein kinase A; DMEM, Dulbecco's modified Eagle's medium; [cAMP]_i, intracellular cAMP concentration; Y27632, R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide; KT5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester.

Address correspondence to: Dr. Masahiro Oike, Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. E-mail: moike{at}pharmaco.med.kyushu-u.ac.jp

	References

Top Abstract Materials and Methods Results Discussion References

 

Black JL, Roth M, Lee J, Carlin S, and Johnson PR (2001) Mechanisms of airway remodeling: airway smooth muscle. Am J Respir Crit Care Med 164: S63-S66.[Abstract/Free Full Text]

Cerutis DR, Nogami M, Anderson JL, Churchill JD, Romberger DJ, Rennard SI, and Toews ML (1997) Lysophosphatidic acid and EGF stimulate mitogenesis in human airway smooth muscle cells. Am J Physiol 273: L10-L15.[Medline]

Dechert MA, Holder JM, and Gerthoffer WT (2001) p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk. Am J Physiol 281: C123-C132.

Diviani D, Baisamy L, and Appert-Collin A (2006) AKAP-Lbc: a molecular scaffold for the integration of cyclic AMP and Rho transduction pathways. Eur J Cell Biol 85: 603-610.[CrossRef][Medline]

Diviani D, Soderling J, and Scott JD (2001) AKAP-Lbc anchors protein kinase A and nucleates Galpha 12-selective Rho-mediated stress fiber formation. J Biol Chem 276: 44247-44257.[Abstract/Free Full Text]

Ellerbroek SM, Wennerberg K, and Burridge K (2003) Serine phosphorylation negatively regulates RhoA in vivo. J Biol Chem 278: 19023-19031.[Abstract/Free Full Text]

Goncharova EA, Billington CK, Irani C, Vorotnikov AV, Tkachuk VA, Penn RB, Krymskaya VP, and Panettieri RA Jr (2003) Cyclic AMP-mobilizing agents and glucocorticoids modulate human smooth muscle cell migration. Am J Respir Cell Mol Biol 29: 19-27.[Abstract/Free Full Text]

Govindaraju V, Michoud MC, Al-Chalabi M, Ferraro P, Powell WS, and Martin JG (2006) Interleukin-8: Novel roles in human airway smooth muscle cell contraction and migration. Am J Physiol 291: C957-C965.[CrossRef]

Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, and Gerthoffer WT (1999) A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 274: 24211-24219.[Abstract/Free Full Text]

Irani C, Goncharova EA, Hunter DS, Walker CL, Panettieri RA, and Krymskaya VP (2002) Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration. Am J Physiol 282: L854-L862.

John M, Hirst SJ, Jose PJ, Robichaud A, Berkman N, Witt C, Twort CH, Barnes PJ, and Chung KF (1997) Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids. J Immunol 158: 1841-1847.[Abstract]

Kaibuchi K, Kuroda S, and Amano M (1999) Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 68: 459-486.[CrossRef][Medline]

Kimura C, Oike M, Koyama T, and Ito Y (2001) Alterations of Ca²⁺ mobilizing properties in migrating endothelial cells. Am J Physiol 281: H745-H754.

Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, and Reusch JE (2001) cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration. J Biol Chem 276: 46132-46141.[Abstract/Free Full Text]

Koyama T, Oike M, and Ito Y (2001) Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells. J Physiol 532: 759-769.[Abstract/Free Full Text]

Kranenburg O, Poland M, Gebbink M, Oomen L, and Moolenaar WH (1997) Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA. J Cell Sci 110: 2417-2427.[Abstract]

Krymskaya VP, Goncharova EA, Ammit AJ, Lim PN, Goncharov DA, Eszterhas A, and Panettieri RA, Jr (2005) Src is necessary and sufficient for human airway smooth muscle cell proliferation and migration. FASEB J 19: 428-430.[Abstract/Free Full Text]

Lang P, Gesbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M, and Bertoglio J (1996) Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J 15: 510-519.[Medline]

Madison JM (2003) Migration of airway smooth muscle cells. Am J Respir Cell Mol Biol 29: 8-11.[Free Full Text]

McIntyre TM, Pontsler AV, Silva AR, St Hilaire A, Xu Y, Hinshaw JC, Zimmerman GA, Hama K, Aoki J, Arai H, et al. (2003) Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARgamma agonist. Proc Natl Acad Sci USA 100: 131-136.[Abstract/Free Full Text]

Moolenaar WH (1995) Lysophosphatidic acid signalling. Curr Opin Cell Biol 7: 203-210.[CrossRef][Medline]

Mukai M, Nakamura H, Tatsuta M, Iwasaki T, Togawa A, Imamura F, and Akedo H (2000) Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP-elevating agents via a Rho-dependent pathway. FEBS Lett 484: 69-73.[CrossRef][Medline]

Mukhina S, Stepanova V, Traktouev D, Poliakov A, Beabealashvilly R, Gursky Y, Minashkin M, Shevelev A, and Tkachuk V (2000) The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain: characterization of interactions and contribution to chemotaxis. J Biol Chem 275: 16450-16458.[Abstract/Free Full Text]

Newby AC and Zaltsman AB (2000) Molecular mechanisms in intimal hyperplasia. J Pathol 190: 300-309.[CrossRef][Medline]

O'Byrne PM, and Inman MD (2003) Airway hyperresponsiveness. Chest 123: 411S-416S.[CrossRef][Medline]

Parameswaran K, Cox G, Radford K, Janssen LJ, Sehmi R, and O'Byrne PM (2002) Cysteinyl leukotrienes promote human airway smooth muscle migration. Am J Respir Crit Care Med 166: 738-742.[Abstract/Free Full Text]

Ridley AJ (2001) Rho GTPases and cell migration. J Cell Sci 114: 2713-2722.[Abstract/Free Full Text]

Sakai J, Oike M, Hirakawa M, and Ito Y (2003) Theophylline and cAMP inhibit lysophosphatidic acid-induced hyperresponsiveness of bovine tracheal smooth muscle cells. J Physiol 549: 171-180.[Abstract/Free Full Text]

Solway J and Fredberg JJ (1997) Perhaps airway smooth muscle dysfunction contributes to asthmatic bronchial hyperresponsiveness after all. Am J Respir Cell Mol Biol 17: 144-146.[Free Full Text]

Stewart AG (2001) Airway wall remodelling and hyperresponsiveness: modelling remodelling in vitro and in vivo. Pulm Pharmacol Ther 14: 255-265.[CrossRef][Medline]

Toews ML, Ediger TL, Romberger DJ, and Rennard SI (2002) Lysophosphatidic acid in airway function and disease. Biochim Biophys Acta 1582: 240-250.[Medline]

Toews ML, Ustinova EE, and Schultz HD (1997) Lysophosphatidic acid enhances contractility of isolated airway smooth muscle. J Appl Physiol 83: 1216-1222.[Abstract/Free Full Text]

Whitehead IP, Campbell S, Rossman KL, and Der CJ (1997) Dbl family proteins. Biochim Biophys Acta 1332: F1-F23.[Medline]

Wilson J (2000) The bronchial microcirculation in asthma. Clin Exp Allergy 30: 51-53.[CrossRef][Medline]

Xie Y, Gibbs TC, and Meier KE (2002) Lysophosphatidic acid as an autocrine and paracrine mediator. Biochim Biophys Acta 1582: 270-281.[Medline]

This article has been cited by other articles:

				L. Chen, J. J. Zhang, and X.-Y. Huang cAMP Inhibits Cell Migration by Interfering with Rac-induced Lamellipodium Formation J. Biol. Chem., May 16, 2008; 283(20): 13799 - 13805. [Abstract] [Full Text] [PDF]

				C. Zhao, M. J. Fernandes, G. D. Prestwich, M. Turgeon, J. Di Battista, T. Clair, P. E. Poubelle, and S. G. Bourgoin Regulation of Lysophosphatidic Acid Receptor Expression and Function in Human Synoviocytes: Implications for Rheumatoid Arthritis? Mol. Pharmacol., February 1, 2008; 73(2): 587 - 600. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.118042v1 321/3/1102    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirakawa, M. Articles by Oike, M. Search for Related Content PubMed PubMed Citation Articles by Hirakawa, M. Articles by Oike, M.