Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M409739200 on November 16, 2004

J. Biol. Chem., Vol. 280, Issue 4, 3112-3120, January 28, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/4/3112    most recent
M409739200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dronadula, N.
Right arrow Articles by Rao, G. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dronadula, N.
Right arrow Articles by Rao, G. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

STAT-3-dependent Cytosolic Phospholipase A2 Expression Is Required for Thrombin-induced Vascular Smooth Muscle Cell Motility*

Nagadhara Dronadula, Zhimin Liu, Chunmei Wang, Huiqing Cao, and Gadiparthi N. Rao{ddagger}

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, August 24, 2004 , and in revised form, October 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A2 (cPLA2) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122–46128). In order to learn more about the STAT-3-cPLA2 axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA2 in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA2 blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA2 also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation is believed to be an initiative event in the pathogenesis of vessel wall diseases (1, 2). The dysfunctional endothelial cells and inflammatory cells at the site of vascular injury produce a large number of molecules with a broad spectrum of biological activities (1–3). A majority of these molecules are either mitogenic, motogenic, or both to vascular smooth muscle cells (VSMC)1 (4–8). Thus, the availability of these substances at the site of vascular injury provides a permissive milieu for VSMC dedifferentiation (9). The dedifferentiated VSMC acquire their embryonic synthetic noncontractile phenotype and contribute to the progression of lesions such as restenosis after percutaneous transluminal angioplasty via their migration from media to intima and multiplication in intima (10). Several studies have reported that inhibition of expression of molecules that are produced at the site of vascular injury or suppression of their biological activities ameliorates the lesion progression (11, 12). As several molecules are involved in the pathogenesis of arterial wall lesions, identifying the signaling mechanisms that are common to many such factors may eventually lead to the development of better therapeutic agents against these vascular diseases.

Janus-activated kinases are a group of nonreceptor tyrosine kinases that, via tyrosine phosphorylation, modulate the activities of a group of transcriptional factors, namely signal transducers and activators of transcription (STATs) (13, 14). A body of accumulating evidence indicates that STATs play an important role in the regulation of cell proliferation and differentiation (15–17). Arachidonic acid, a polyunsaturated fatty acid, is an important component of membrane phospholipids and is released acutely in response to a variety of agents, including cytokines, growth factors, hormones, and oxidants (18–22). Upon release, it is either metabolized via the cyclooxygenase, lipoxygenase, or cytochrome P450 monooxygenase pathways producing prostaglandins, hydroperoxyeicosatetraenoic acids, and epoxyeicosatrienoic acids, respectively, or is reincorporated into membrane phospholipids via esterification involving arachidonoyl-CoA synthase and arachidonoyl-lysophospholipid transferase (18, 23). Arachidonic acid and its metabolites are also involved in the regulation of vital physiological processes such as vascular tone (23, 24). In addition, these lipid molecules have been reported to mediate intracellular signaling events in response to a number of external cues (25–30). Arachidonic acid and its metabolites have also been shown to play a role in cell survival and proliferation (31–35). Among the large family of PLA2s identified thus far, cPLA2 appears to be one of the major sources of arachidonic acid release and therefore the production of its metabolites, eicosanoids, in response to various agonists in different cell types (36–38). Recently, we have reported that Jak/STAT-dependent expression of cPLA2 is required for PDGF-BB-induced VSMC proliferation and migration (39, 40). Thrombin, a serine protease, plays an indispensable role in hemostasis (41). In addition to its role in platelet activation and blood clot formation, thrombin influences proliferation and migration in many cell types including VSMC (42–45). The signaling events underlying the mitogenic responses of thrombin were fairly studied, particularly in the context of a requirement for transactivation of receptor tyrosine kinases such as epidermal growth factor receptor and insulin-like growth factor 1 receptor, activation of nonreceptor tyrosine kinases such as Src and Jak-2, and serine/threonine kinases such as mitogen-activated protein kinases (42–47). However, the signaling mechanisms by which thrombin stimulates cell motility are less understood. To gain insight into the motogenic signaling events of GPCR agonists, here we have studied the role of STAT-3 in thrombin-induced VSMC migration. The major finding of the present study is that thrombin induces VSMC motility via activation of STAT-3 targeting the induction of expression of cPLA2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Aprotinin, dithiothreitol, HEPES, phenylmethylsulfonyl fluoride, sodium orthovanadate, sodium deoxycholate, leupeptin, and thrombin were purchased from Sigma. Arachidonic acid was bought from Cayman Chemicals (Ann Arbor, MI). Phospho-specific anti-STAT-3 (9131S) antibodies were procured from Cell Signaling Technology (Beverly, MA). Anti-cPLA2 antibodies (SC-454), anti-p53 antibodies (SC-6243), anti-STAT-3 antibodies (SC-482), and consensus STAT-3 binding oligonucleotide (5'-GATCCTTCTGGGAATTCCTAGATC-3' and 3'-CTAGGAAGACCCTTAAGGATCTAG-5') (SC-2571) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). T4 polynucleotide kinase was purchased from Promega (Madison, WI). TRIzol reagent was bought from Invitrogen. Nytran membrane was purchased from Schleicher & Schuell. [{alpha}-32P]dCTP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Ready-to-Go DNA labeling beads and [{gamma}-32 P]ATP (3000 Ci/mmol) were from Amersham Biosciences. Construction of pFS3DM, a dominant negative STAT-3 plasmid, and pcDNA3-cPLA2, a cPLA2 expression plasmid, were described previously (17, 48).

Cell Culture—VSMC were isolated from the thoracic aortae of 200–300 g male Sprague-Dawley rats by enzymatic dissociation as described earlier (39). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37 °C in a humidified 95% air and 5% CO2 atmosphere. Cells were quiesced by incubating in DMEM containing 0.1% calf serum for 72 h and used to perform the experiments unless otherwise stated.

Cell Motility—VSMC motility was measured by cell wounding assay (49). Quiescent confluent monolayers of VSMC were wounded with a sterile pipette tip to generate a cell-free gap of ~1 mm width, and the wound location in the culture dish was marked. Cells were washed, and fresh serum-free DMEM was added and photographed to record the wound width at 0 h. To prevent replicative DNA synthesis, hydroxyurea was added to the medium to a final concentration of 5 mM just before the addition of agonist. Twenty four hours after the appropriate treatments, photographs were taken again at the marked wound location. Cell migration was measured using the NIH image 1.62 program, and the cell motility was expressed as distance migrated in micrometer units. To test the effect of dominant negative mutant of STAT-3 on thrombin-induced motility, cells were first transfected with FS3DM or an empty vector for 48 h and quiesced before they were subjected to agonist-induced motility.

Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared from treated or untreated VSMC as described previously (39). The protein content of the nuclear extracts was determined by using a Micro BCATM Protein Assay Reagent kit (Pierce). Protein-DNA complexes were formed by incubating 5 µg of nuclear protein in a total volume of 20 µl consisting of 15 mM HEPES, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 µg of bovine serum albumin, 2 µg of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32P-labeled oligonucleotide probe for 30 min on ice. The protein-DNA complexes were resolved by electrophoresis on a 4% polyacrylamide gel using 1x Tris-glycine/EDTA buffer (25 mM Tris-HCl, pH 8.5, 200 mM glycine, 0.1 mM EDTA). Double-stranded oligonucleotides were labeled with [{gamma}-32P]ATP using the T4 polynucleotide kinase kit (Promega) following the supplier's protocol.

Northern Blot Analysis—After appropriate treatments, total cellular RNA was isolated using TRIzol reagent following the manufacturer's protocol (Invitrogen). An equal amount of total cellular RNA (20 µg) from control and treated cells was size-fractionated on 1% agarose gel in 25 mM MOPS buffer, pH 7.8, containing 1 mM EDTA and 2% formaldehyde. RNA was transferred to a Nytran membrane (Schleicher & Schuell) and cross-linked to the membrane using ultraviolet irradiation (Stratalinker, Stratagene, La Jolla, CA). The 2.1-kb rat cPLA2 cDNA fragment isolated by digestion of pcDNA3-cPLA2 expression plasmid with EcoRI was labeled with [{alpha}-32P]dCTP using Ready-to-Go DNA labeling beads (Amersham Biosciences). After a 4-h prehybridization in 50% formamide, 5x SSC, 5x Denhardt's, 50 mM sodium phosphate, pH 6.5, and 250 µg/ml of sheared salmon sperm DNA at 42 °C, the Nytran membrane was hybridized in the above buffer containing 10% dextran sulfate and 1 x 106 cpm/ml of rat cPLA2 cDNA probe for 16 h at 42 °C. The Nytran membrane was washed three times for 15 min each at room temperature in 2x SSC containing 0.2% SDS and two times for 30 min each at 60 °C in 0.1x SSC containing 0.1% SDS. The membrane was exposed to an x-ray film (Hawkins X-Ray film) with an intensifying screen at -80 °C for 2 days. The membrane was re-probed with [{alpha}-32P]dCTP-labeled 18 S rDNA probe for lane loading control. Densitometric analysis of autoradiograms exposed in the linear range of film density was made using the NIH image 1.62 program.

Western Blot Analysis—After appropriate treatments, VSMC were rinsed with cold phosphate-buffered saline and lysed in 250 µl of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate) on ice for 20 min. The cell lysates were scraped into 1.5-ml Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min at 4 °C. Cell extracts containing an equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Biosciences). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected by using a chemiluminescence reagent kit (Amersham Biosciences).

Transient Transfection and CAT Assay—VSMC were plated evenly onto 100-mm dishes and grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At 50–80% confluence, medium was replaced with DMEM containing 0.1% calf serum, and cells were transfected with pSIE-CAT plasmid using Lipofectamine plus reagent according to the manufacturer's instructions (Invitrogen). Thirty hours after transfection, VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. Wherever the effect of dominant negative STAT-3 mutant was tested on agonist-induced CAT activity, cells were co-transfected with pSIE-CAT along with and without FS3DM or empty vector plasmid DNA. VSMC extracts were normalized for protein and assayed for CAT activity using [14C]chloramphenicol and acetyl coenzyme A as substrates. The substrate and products were extracted with ethyl acetate, separated by TLC, and subjected to autoradiography.

Statistics—All the experiments were repeated three times with similar results. Data are presented as mean ± S.D. The treatment effects were analyzed by Student's t test. p values < 0.05 were considered to be statistically significant. In the case of CAT activity, EMSA, Northern blotting, and Western blotting, one representative set of data is shown.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To understand the motogenic signaling events of thrombin, we first determined its dose-response effect on VSMC motility. A cell-free gap was generated in a monolayer of quiescent VSMC as described under "Materials and Methods." Cells were then treated with and without various doses of thrombin for 24 h, and cell motility was measured. As shown in Fig. 1, thrombin induced VSMC motility in a dose-dependent manner. Maximum VSMC motility was observed in response to 0.5 units/ml of thrombin. To test the role of STATs in GPCR agonist-induced VSMC motility, we tested the effect of thrombin on STAT-3 activation. Quiescent VSMC were treated with and without thrombin (0.5 units/ml) for various times, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for tyrosine phosphorylation of STAT-3 using its phospho-specific antibodies. Thrombin stimulated tyrosine phosphorylation of STAT-3 in a time-dependent manner with a maximum effect of about 4-fold increase at 10 min and reaching basal levels by 60 min (Fig. 2). To find whether thrombin-induced STAT-3 tyrosine phosphorylation leads to its translocation from the cytoplasm to the nucleus, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for 10 min, and the cytoplasmic and nuclear extracts were prepared. An equal amount of protein from the cytoplasmic and nuclear extracts of control and thrombin-treated cells was analyzed by Western blotting for tyrosine phosphorylation of STAT-3 as described above. First, the level of tyrosine-phosphorylated STAT-3 was found to be higher in the nuclear fraction than in the cytoplasmic fraction of both control and thrombin-treated cells (Fig. 3). Second, the level of tyrosine-phosphorylated STAT-3 in the nuclear fraction of thrombin-treated cells was at least 2-fold higher than its amount in the nuclear fraction of control cells (Fig. 3). In addition, forced expression of FS3DM, a dominant negative mutant of STAT-3, reduced the tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus in both thrombin-treated and untreated cells. Furthermore, and as expected, in cells transfected with FS3DM, the total amount of STAT-3 was found to be 2-fold higher than in nontransfected cells. The total STAT-3 levels were found to be much lower in nuclear fractions as compared with cytoplasmic fractions, and these levels correlated with the amounts of its phosphorylated form. The relative purity of nuclear preparations was confirmed by re-probing these Western blots for p53 using its specific antibodies. As evident from Fig. 3, p53 was detected only in the nuclear but not in the cytoplasmic fractions.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1.
Thrombin induces VSMC motility in a dose-dependent manner. A cell-free gap was made in a monolayer of quiescent VSMC and treated with and without various doses of thrombin for 24 h, and cell motility was measured using the NIH Image 1.62 program. *, p < 0.01 versus control.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2.
Thrombin stimulates tyrosine phosphorylation of STAT-3 in a time-dependent manner in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for pSTAT-3 using its phospho-specific antibodies. As a lane loading control, the blot was re-probed with anti-STAT-3 antibodies.

 



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3.
FS3DM, a dominant negative STAT-3 mutant, suppresses thrombin-induced tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus. Quiescent VSMC that were transfected with and without FS3DM or an empty vector were quiesced and treated with and without thrombin (0.5 units/ml) for 10 min, and the cytoplasmic and nuclear extracts were prepared. An equal amount of protein from the cytoplasmic and nuclear extracts of VSMC that were subjected to various treatments or left untreated was analyzed by Western blotting for pSTAT-3 using its phospho-specific antibodies. As a lane loading control and to check the relative purity of nuclear extracts, the blots were re-probed sequentially with anti-STAT-3 and anti-p53 antibodies.

 
Upon tyrosine phosphorylation, STATs undergo either homo- or heterodimerization and translocate to the nucleus where they bind to their consensus DNA-binding sequences present in the promoter regions of genes and influence their transcription (13, 50). To find whether the increases in its tyrosine phosphorylation correlate with its transcriptional activation, the STAT-3-DNA binding activity was measured. Quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and nuclear extracts were prepared. An equal amount of nuclear protein from control and each treatment was analyzed by EMSA for STAT-3-DNA binding activity using 32P-labeled consensus STAT-3-DNA binding oligonucleotide as a probe. Thrombin increased STAT3-DNA binding activity in a time-dependent manner with a maximum effect at 4 h (Fig. 4A). To confirm that the increased STAT-3-DNA binding activity leads to an increase in its transactivation activity, VSMC were transiently transfected with a STAT-3-dependent reporter plasmid, pSIE-CAT, quiesced, and treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. Cell extracts normalized for protein were assayed for CAT activity. Consistent with its effect on DNA binding activity, thrombin induced STAT-3-dependent CAT activity in a time-dependent manner as well (Fig. 4B). A maximum 2-fold increase in STAT-3-dependent CAT activity was observed at a 4-h thrombin treatment versus control (Fig. 4C). To confirm these results, we next tested the effect of a dominant negative mutant of STAT-3, FS3DM. VSMC were transfected with and without FS3DM or an empty vector, quiesced, and treated with and without thrombin (0.5 units/ml) for 2 h, and nuclear extracts were prepared and analyzed for STAT-3-DNA binding activity as described above. As shown in Fig. 5A, forced expression of FS3DM significantly blocked thrombin-induced STAT-3-DNA binding activity. To determine the effect of FS3DM on STAT-3-dependent reporter gene activity, VSMC were co-transfected with pSIE-CAT along with and without FS3DM or an empty vector, quiesced, and treated with and without thrombin for 4 h, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed for CAT activity as described above. FS3DM also blocked thrombin-induced STAT-3-dependent CAT activity (Fig. 5, B and C). In order to understand the role of STAT-3 in thrombin-induced VSMC motility, we next tested the effect of FS3DM. Forced expression of FS3DM significantly prevented thrombin-induced VSMC motility (Fig. 6).



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 4.
Thrombin induces STAT-3-DNA binding and reporter gene activities in a time-dependent manner. A, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and nuclear extracts were prepared. An equal amount of nuclear protein from control and each treatment was incubated with 100,000 cpm of 32P-labeled consensus STAT-3 binding oligonucleotide probe, and the protein-DNA complexes were separated by EMSA and subjected to autoradiography. B, VSMC that were transfected with a STAT-3-dependent reporter plasmid, pSIE-CAT, were quiesced, treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were analyzed for CAT activity using [14C]chloramphenicol and acetyl coenzyme A as substrates. The substrate and products were extracted with ethyl acetate, separated by TLC, and subjected to autoradiography. C, the bar graph represents the quantitative analysis of three independent experiments for CAT activity. *, p < 0.01 versus control.

 



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 5.
FS3DM, a dominant negative mutant of STAT-3, reduces thrombin-induced STAT-3-DNA binding and reporter gene activities. A, VSMC that were transfected with and without FS3DM or an empty vector were quiesced and treated with and without thrombin (0.5 units/ml) for 2 h, and nuclear extracts were prepared. An equal amount of nuclear protein from control and each treatment was incubated with 100,000 cpm of 32P-labeled consensus STAT-3 binding oligonucleotide probe, and the protein-DNA complexes were separated by EMSA and subjected to autoradiography. B, VSMC that were cotransfected with a STAT-3-dependent reporter plasmid, pSIE-CAT, along with and without FS3DM or an empty vector were quiesced and treated with and without thrombin (0.5 units/ml) for 4 h, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were analyzed for CAT activity using [14C]chloramphenicol and acetyl coenzyme A as substrates. The substrate and products were extracted with ethyl acetate, separated by TLC, and subjected to autoradiography. C, the bar graph represents the quantitative analysis of three independent experiments for CAT activity. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment alone.

 



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 6.
Blockade of STAT-3 activation prevents thrombin-induced VSMC motility. VSMC that were transfected with and without FS3DM, a dominant negative STAT-3 mutant, or an empty vector were quiesced, and a cell-free gap was made. Cells were then treated with and without thrombin (0.5 units/ml) for 24 h, and cell motility was measured using the NIH image 1.62 program. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment alone.

 
It was reported that arachidonic acid and its eicosanoid metabolites play an important role in cell migration (51–54). In addition, arachidonic acid, the precursor for eicosanoids, and other mitogenically active lipids such as phosphatidic acid have been shown to activate GTPases via inhibition of GTPase-activating proteins (29, 30, 55, 56). GTPases play an essential role in cell proliferation and migration (57). Since cPLA2 plays a predominant role in arachidonic acid release in response to a variety of bioactive agents, including growth factors (27, 36–38), we envisioned that STAT-3 signaling may be mediating cell migration via induction of expression of cPLA2. To test this possibility, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for various times, and total cellular RNA was isolated. An equal amount of RNA from control and thrombin-treated cells was analyzed by Northern blotting for cPLA2 by using 32P-labeled rat cPLA2 cDNA probe. Thrombin induced the expression of cPLA2 mRNA in a time-dependent manner with a maximum effect at 8 h. Thrombin had no significant effect on 18 S RNA levels (Fig. 7A). To find out whether the increased expression of cPLA2 mRNA correlates with an increase in its protein levels, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and thrombin-treated cells was analyzed for cPLA2 using its specific antibodies. Consistent with its effect on cPLA2 mRNA expression, thrombin also induced the production of cPLA2 at protein levels (Fig. 7B). To understand the role of STAT-3 in thrombin-induced cPLA2 expression, we next studied the effect of FS3DM. As shown in Fig. 8, A and B, forced expression of FS3DM blocked thrombin-induced expression of cPLA2 at both mRNA and protein levels. Next we examined the role of cPLA2 in thrombin-induced VSMC motility using a pharmacological approach. Use of methyl arachidonyl fluorophosphonate (MAFP) and palmitoyl trifluoromethyl ketone, two structurally different and specific inhibitors of cPLA2 (38, 58), significantly blocked thrombin-induced VSMC motility (Fig. 9). If STAT-3 signaling mediates thrombin-induced VSMC motility via induction of expression of cPLA2, then one would expect that exogenous addition of arachidonic acid rescues thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. To test this, a cell-free gap was made in a monolayer of quiescent VSMC that were forced to express FS3DM and treated with and without thrombin (0.5 units/ml) in the presence and absence of the indicated concentrations of exogenously added arachidonic acid for 24 h, and cell motility was measured. As shown in Fig. 10A, arachidonic acid at 5 µM concentration rescued thrombin-induced VSMC motility from inhibition by FS3DM. Arachidonic acid at 10 µM concentration over a 24-h period caused mild toxicity to VSMC and, therefore, was found to be less effective in rescuing thrombin-induced VSMC migration from inhibition by FS3DM. To confirm this result further, VSMC were co-transfected with and without FS3DM or an empty vector in combination with and without an expression plasmid for rat cPLA2, pcDNA3-cPLA2, and quiesced. A cell-free gap was then made in the monolayer and treated with and without thrombin (0.5 units/ml) for 24 h, and cell motility was measured. Forced expression of rat cPLA2 rescued thrombin-induced VSMC motility from inhibition by the dominant negative STAT-3 mutant, FS3DM (Fig. 10B).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 7.
Thrombin induces the expression of cPLA2 in a time-dependent manner. A, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and total cellular RNA was isolated. An equal amount of RNA from control and each treatment was analyzed by Northern blotting for cPLA2 using 32P-labeled rat cPLA2 cDNA probe. The blot was re-probed with 32P-labeled 18 S rDNA probe for the purpose of lane loading control. The bar graph represents the quantitative analysis of two independent Northern blot experiments. B, quiescent VSMC were treated with and without thrombin (0.5 units/ml) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for cPLA2 using its specific antibodies. The bar graph represents the quantitative analysis of three independent Western blot experiments. *, p < 0.01 versus control.

 



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 8.
FS3DM, a dominant negative STAT-3 mutant, inhibits thrombin-induced expression of cPLA2. A, VSMC that were transfected with and without FS3DM or an empty vector were quiesced and treated with and without thrombin (0.5 units/ml) for 8 h, and total cellular RNA was isolated. An equal amount of RNA from control and each treatment was analyzed by Northern blotting for cPLA2 using 32P-labeled rat cPLA2 cDNA probe. The blot was re-probed with 32P-labeled 18 S rDNA probe for the purpose of lane loading control. The bar graph represents the quantitative analysis of three independent Northern blot experiments. B, VSMC that were transfected with and without FS3DM or empty vector were quiesced and treated with and without thrombin (0.5 units/ml) for 8 h, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for cPLA2 using its specific antibodies. Similar results were obtained in three independent experiments. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment alone.

 



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 9.
MAFP and palmitoyl trifluoromethyl ketone, selective inhibitors of cPLA2, reduce thrombin-induced VSMC motility. A cell-free gap was made in a monolayer of quiescent VSMC and treated with and without thrombin (0.5 units/ml) in the presence and absence of MAFP (10 µM) or palmitoyl trifluoromethyl ketone (PACOCF3) (10 µM) for 24 h, and cell motility was measured using the NIH image 1.62 program. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment alone.

 



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 10.
Exogenous addition of arachidonic acid or forced expression of cPLA2 rescued thrombin-induced VSMC motility from inhibition by a dominant negative STAT-3 mutant, FS3DM. A, VSMC were transfected first with and without FS3DM or empty vector and then quiesced. A cell-free gap was made in the monolayer of quiescent VSMC and then treated with and without thrombin (0.5 units/ml) in the presence and absence of the indicated concentrations of arachidonic acid (AA) for 24 h, and cell motility was measured using the NIH image 1.62 program. B, VSMC that were co-transfected with a dominant negative STAT-3 mutant, FS3DM or empty vector, along with and without an expression plasmid for rat cPLA2, pcDNA3-cPLA2, and quiesced were treated with and without thrombin (0.5 units/ml) for 24 h, and cell motility was measured as described above. *, p < 0.01 versus control; **, p < 0.01 versus thrombin treatment alone; ***, p < 0.01 versus FS3DM + thrombin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The important finding of the present study is that thrombin, a GPCR agonist, induces VSMC motility via STAT-3-dependent induction of expression of cPLA2. This conclusion is supported by the following observations. 1) Thrombin stimulated tyrosine phosphorylation of STAT-3 in a time-dependent manner. 2) Thrombin also increased STAT-3-DNA binding and reporter gene activities. 3) FS3DM, a dominant negative STAT-3 mutant (17), inhibited thrombin-induced STAT-3 tyrosine phosphorylation, its translocation from the cytoplasm to the nucleus, and DNA binding and reporter gene activities. 4) Thrombin stimulated VSMC motility in a dose-dependent manner, and FS3DM suppressed this response. 5) Thrombin induced cPLA2 expression in a time-dependent manner both at mRNA and protein levels. 6) Blockade of STAT-3 activation by its dominant negative mutant significantly reduced thrombin-induced cPLA2 expression both at mRNA and protein levels. 7) Pharmacological inhibition of cPLA2 blocked thrombin-induced VSMC motility. 8) Exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by the blockade of STAT-3 activation. 9) Forced expression of rat cPLA2 also had overcome the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. In addition to their role in cell proliferation and differentiation (15–17), emerging evidence indicates the involvement of STATs in the regulation of cell migration (59, 60). Although the structure and function relationship of these transcriptional factors were well studied (13, 14), the mechanisms of their action in the regulation of cell migration and proliferation are largely unclear. In this regard, the present study provides evidence for a role of cPLA2 as one of the distal molecules in STAT-3-mediated cell migration. Analysis of both human and rat cPLA2 promoters for regulatory elements also revealed the presence of several binding sequences for STATs (61, 62). The finding that a dominant negative mutant of STAT-3 suppressed thrombin-induced expression of cPLA2 both at mRNA and protein levels and the presence of STAT regulatory elements in its promoter further support a role for cPLA2 in cellular responses such as cell motility mediated by STAT transcriptional factors.

It was reported that arachidonic acid and its eicosanoid metabolites play a role in cell migration (51–54). Arachidonic acid and its eicosanoid metabolites have also been shown to activate GTPases via inhibition of GTPase-activating proteins (29, 30, 55, 56). GTPases play an essential role in cell migration (57). Based on these observations, it is presumable that STAT-3-mediated cPLA2 expression may be involved in a sustained arachidonic acid release producing eicosanoids that in turn promote VSMC chemotaxis. A large body of evidence indicates that cPLA2 mediates the release of arachidonic acid and, thereby, eicosanoid production in response to a variety of bioactive agents (18–22). In earlier studies, we have reported that STAT-3 mediates PDGF-BB-induced VSMC proliferation and migration via induction of expression of cPLA2 and arachidonic acid release (39, 40). Arachidonic acid, while serving as a precursor for the production of biologically important lipid mediators, is also a crucial component for membrane phospholipid remodeling. PLA2s via production of arachidonic acid and lipid mediators have been reported to play a role in membrane phospholipid remodeling (63, 64). Plasma membrane protrusion, retraction, and contraction forces that are essential for cell motility involve membrane phospholipid remodeling. In this regard, it is tempting to speculate that STAT-3-dependent cPLA2 expression may be involved in membrane phospholipid remodeling during cell motility. Future studies are required to test the role of STAT-3-cPLA2 signaling axis in thrombin-induced cytoskeleton and membrane phospholipid remodeling.

In summary, the present study demonstrates that STAT-3 mediates thrombin-induced motility in VSMC via targeting the induction of expression of cPLA2.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL64165 and HL69908 (to G. N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7321; Fax: 901-448-7126; E-mail: grao{at}physio1.utmem.edu.

1 The abbreviations used are: VSMC, vascular smooth muscle cells; cPLA2, cytosolic phospholipase A2; GPCR, G protein-coupled receptor; PDGF-BB, platelet-derived growth factor-BB; STAT, signal transducers and activators of transcription; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; CAT, chloramphenicol acetyltransferase; PDGF, platelet-derived growth factor; EMSA, electrophoretic mobility shift assay; MAFP, methyl arachidonyl fluorophosphonate. Back


   ACKNOWLEDGMENTS
 
We are thankful to Drs. Ralph A. Bradshaw and Z. Alex Ma for providing us with FS3DM, a dominant negative STAT-3 plasmid, and pcDNA3-cPLA2, an expression plasmid for cPLA2, respectively. We are also grateful to Laxmisilpa Gadiparthi for typing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ross, R. (1999) N. Engl. J. Med. 340, 115-126[Free Full Text]
  2. Hansson, G. K., Libby, P., Schonbeck, U., and Yan, Z. Q. (2002) Circ. Res. 91, 281-291[Abstract/Free Full Text]
  3. Berk, B. C. (2001) Physiol. Rev. 81, 999-1030[Abstract/Free Full Text]
  4. Jawien, A., Bowen-Pope, D. F., Lindner, V., Schwartz, S. M., and Clowes, A. W. (1992) J. Clin. Investig. 89, 507-511[Medline] [Order article via Infotrieve]
  5. Kalmes, A., Vesti, B. R., Daum, G., Abraham, J. A., and Clowes, A. W. (2000) Circ. Res. 87, 92-98[Abstract/Free Full Text]
  6. Goetze, S., Xi, X. P., Kawano, Y., Kawano, H., Fleck, E., Hsueh, W. A., and Law, R. E. (1999) Hypertension 33, 183-189[Abstract/Free Full Text]
  7. Duan, C., Bauchat, J. R., and Hsieh, T. (2000) Circ. Res. 86, 15-23[Abstract/Free Full Text]
  8. Chai, Y. C., Howe, P. H., Dicorleto, P. E., and Chisolm, G. M. (1996) J. Biol. Chem. 271, 17791-17797[Abstract/Free Full Text]
  9. Kumar, M. S., and Owens, G. K. (2003) Arterioscler. Thromb. Vasc. Biol. 23, 737-747[Abstract/Free Full Text]
  10. Regan, C. P., Adam, P. J., Madsen, C. S., and Owens, G. K. (2000) J. Clin. Investig. 106, 1139-1147[Medline] [Order article via Infotrieve]
  11. Ferns, G. A., Raines, E. W., Sprugel, K. H., Motani, A. S., Reidy, M. A., and Ross, R. (1991) Science 253, 1129-1132[Abstract/Free Full Text]
  12. Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743[Abstract/Free Full Text]
  13. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421[Abstract/Free Full Text]
  14. Horvath, C. M., Wen, Z., and Darnell, J. E., Jr. (1995) Genes Dev. 9, 984-994[Abstract/Free Full Text]
  15. Fukada, T., Ohtani, T., Yoshida, Y., Shirogane, T., Nishida, K., Nakajima, K., Hibi, M., and Hirano, T. (1998) EMBO J. 17, 6670-6677[CrossRef][Medline] [Order article via Infotrieve]
  16. Nosaka, T., Kawashima, T., Misawa, K., Ikuta, K., Mui, A. L., and Kitamura, T. (1999) EMBO J. 18, 4754-4765[CrossRef][Medline] [Order article via Infotrieve]
  17. Wu, Y. Y., and Bradshaw, R. A. (2000) J. Biol. Chem. 275, 2147-2156[Abstract/Free Full Text]
  18. Piomelli, D. (1993) Curr. Opin. Cell Biol. 5, 274-280[CrossRef][Medline] [Order article via Infotrieve]
  19. Dethlefsen, S. M., Shepro, D., and D'Amore, P. A. (1994) Exp. Cell Res. 212, 262-273[CrossRef][Medline] [Order article via Infotrieve]
  20. Gronich, J., Konieczkowski, M., Gelb, M. H., Nemenoff, R. A., and Sedor, J. R. (1994) J. Clin. Investig. 93, 1224-1233[Medline] [Order article via Infotrieve]
  21. Rao, G. N., Lassegue, B., Alexander, R. W., and Griendling, K. K. (1994) Biochem. J. 299, 197-201[Medline] [Order article via Infotrieve]
  22. Rao, G. N., Runge, M. S., and Alexander, R. W. (1995) Biochim. Biophys. Acta 1265, 67-72[Medline] [Order article via Infotrieve]
  23. Smith, W. L. (1989) Biochem. J. 259, 315-324[Medline] [Order article via Infotrieve]
  24. Lin, L., Balazy, M., Pagano, P. J., and Nasjletti, A. (1994) Circ. Res. 74, 197-205[Abstract/Free Full Text]
  25. Graber, M. N., Alfonso, A., and Gill, D. L. (1996) J. Biol. Chem. 271, 883-888[Abstract/Free Full Text]
  26. Rao, G. N., Baas, A. S., Glasgow, W. C., Eling, T. E., Runge, M. S., and Alexander, R. W. (1994) J. Biol. Chem. 269, 32586-32591[Abstract/Free Full Text]
  27. Peppelenbosch, M. P., Tertoolen, L. G. J., Hage, W. J., and deLaat, S. W. (1993) Cell 74, 565-575[CrossRef][Medline] [Order article via Infotrieve]
  28. Fafeur, V., Jiang, Z. P., and Bohlen, P. (1991) J. Cell. Physiol. 149, 277-283[CrossRef][Medline] [Order article via Infotrieve]
  29. Golubic, M., Tanaka, K., Dobrowolski, S., Wood, D., Tsai, M. H., Marshall, M., Tamanoi, F., and Stacey, D. W. (1991) EMBO J. 10, 2897-2903[Medline] [Order article via Infotrieve]
  30. Tsai, M. H., Roudebush, M., Dobrowolski, S., Yu, C. L., Gibbs, J. B., and Stacey, D. W. (1991) Mol. Cell. Biol. 11, 2785-2793[Abstract/Free Full Text]
  31. Glasgow, W. C., Afshari, C. A., Barrett, J. C., and Eling, T. E. (1992) J. Biol. Chem. 267, 10771-10779[Abstract/Free Full Text]
  32. Sellmayer, A., Uedelhoven, W. M., Weber, P. C., and Bonventre, J. V. (1991) J. Biol. Chem. 266, 3800-3807[Abstract/Free Full Text]
  33. Mezentsev, A., Seta, F., Dunn, M. W., Ono, N., Falck, J. R., and Laniado-Schwartzman, M. (2002) J. Biol. Chem. 277, 18670-18676[Abstract/Free Full Text]
  34. Tang, D. G., Chen, Y. Q., and Honn, K. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5241-5246[Abstract/Free Full Text]
  35. Chen, J. K., Capdevila, J., and Harris, R. C. (2001) Mol. Cell. Biol. 21, 6322-6331[Abstract/Free Full Text]
  36. Fujishima, H., Sanchez Mejia, R. O., Bingham, C. O., Lam, B. K., Sapirstein, A., Bonventre, J. V., Austen, K. F., and Arm, J. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4803-4807[Abstract/Free Full Text]
  37. Leslie, C. C. (1997) J. Biol. Chem. 272, 16709-16712[Free Full Text]
  38. Balsinde, J., and Dennis, E. A. (1996) J. Biol. Chem. 271, 6758-6765[Abstract/Free Full Text]
  39. Yellaturu, C. R., and Rao, G. N. (2003) J. Biol. Chem. 278, 9986-9992[Abstract/Free Full Text]
  40. Neeli, I., Liu, Z., Dronadula, N., Ma, Z. A., and Rao, G. N. (2004) J. Biol. Chem. 279, 46122-46128[Abstract/Free Full Text]
  41. Butenas, S., Orfeo, T., Gissel, M. T., Brummel, K. E., and Mann, K. G. (2004) J. Biol. Chem. 279, 22875-22882[Abstract/Free Full Text]
  42. Rao, G. N., and Runge, M. S. (1996) J. Biol. Chem. 271, 20805-20810[Abstract/Free Full Text]
  43. Rao, G. N., Delafontaine, P., and Runge, M. S. (1995) J. Biol. Chem. 270, 27871-27875[Abstract/Free Full Text]
  44. Darmoul, D., Gratio, V., Devaud, H., Lehy, T., and Laburthe, M. (2003) Am. J. Pathol. 162, 1503-1513[Abstract/Free Full Text]
  45. Rauch, B. H., Millette, E., Kenagy, R. D., Daum, G., and Clowes, A. W. (2004) Circ. Res. 94, 340-345[Abstract/Free Full Text]
  46. Marinissen, M. J., Servitja, J. M., Offermanns, S., Simon, M. I., and Gutkind, J. S. (2003) J. Biol. Chem. 278, 46814-46825[Abstract/Free Full Text]
  47. Madamanchi, N. R., Li, S., Patterson, C., and Runge, M. S. (2001) J. Biol. Chem. 276, 18915-18924[Abstract/Free Full Text]
  48. Ma, Z., Ramanadham, S., and Turk, J. (1998) Biochim. Biophys. Acta 1391, 384-400[Medline] [Order article via Infotrieve]
  49. Zhuang, D., Ceacareanu, A. C., Lin, Y., Ceacareanu, B., Dixit, M., Chapman, K. E., Waters, C. M., Rao, G. N., and Hassid, A. (2004) Am. J. Physiol. 286, H2103-H2112
  50. Ehret, G. B., Reichenbach, P., Schindler, U., Horvath, C. M., Fritz, S., Nabholz, M., and Bucher, P. (2001) J. Biol. Chem. 276, 6675-6688[Abstract/Free Full Text]
  51. Ott, V. L., Cambier, J. C., Kappler, J., Marrack, P., and Swanson, B. J. (2003) Nat. Immun. 10, 974-981
  52. Honig, S. M., Fu, S., Mao, X., Yopp, A., Gunn, M. D., Randolph, G. J., and Bromberg, J. S. (2003) J. Clin. Investig. 111, 627-637[CrossRef][Medline] [Order article via Infotrieve]
  53. Stockton, R. A., and Jacobson, B. S. (2001) Mol. Biol. Cell 12, 1937-1956[Abstract/Free Full Text]
  54. Maddox, J. F., Colgan, S. P., Clish, C. B., Petasis, N. A., Fokin, V. V., and Serhan, C. N. (1998) FASEB J. 12, 487-494[Abstract/Free Full Text]
  55. Tsai, M. H., Yu, C. L., Wei, F. S., and Stacey, D. W. (1989) Science 243, 522-526[Abstract/Free Full Text]
  56. Han, J. W., McCormick, F., and Macara, I. G. (1991) Science 252, 576-579[Abstract/Free Full Text]
  57. Etienne-Manneville, S., and Hall, A. (2002) Nature 420, 629-635[CrossRef][Medline] [Order article via Infotrieve]
  58. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) J. Biol. Chem. 270, 445-450[Abstract/Free Full Text]
  59. Silver, D. L., and Montell, D. J. (2001) Cell 107, 831-841[CrossRef][Medline] [Order article via Infotrieve]
  60. Yahata, Y., Shirakata, Y., Tokumaru, S., Yamasaki, K., Sayama, K., Hanakawa, Y., Detmar, M., and Hashimoto, K. (2003) J. Biol. Chem. 278, 40026-40031[Abstract/Free Full Text]
  61. Wu, T., Ikezono, T., Angus, C. W., and Shelhamer, J. H. (1994) Nucleic Acids Res. 22, 5093-5098[Abstract/Free Full Text]
  62. Tay, A., Maxwell, P., Li, Z. G., Goldberg, H., and Skorecki, K. (1994) Biochim. Biophys. Acta 1217, 345-347[Medline] [Order article via Infotrieve]
  63. Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8527-8531[Abstract/Free Full Text]
  64. Serhan, C. N., Haeggstrom, J. Z., and Leslie, C. C. (1996) FASEB J. 10, 1147-1158[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
C.-C. Yu, M.-J. Hsu, M.-L. Kuo, R. F.-C. Chen, M.-C. Chen, K.-J. Bai, M.-C. Yu, B.-C. Chen, and C.-H. Lin
Thrombin-Induced Connective Tissue Growth Factor Expression in Human Lung Fibroblasts Requires the ASK1/JNK/AP-1 Pathway
J. Immunol., June 15, 2009; 182(12): 7916 - 7927.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J.-H. Tsou, K.-Y. Chang, W.-C. Wang, J. T. Tseng, W.-C. Su, L.-Y. Hung, W.-C. Chang, and B.-K. Chen
Nucleolin regulates c-Jun/Sp1-dependent transcriptional activation of cPLA2{alpha} in phorbol ester-treated non-small cell lung cancer A549 cells
Nucleic Acids Res., January 17, 2008; 36(1): 217 - 227.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
V. Kundumani-Sridharan, D. Wang, M. Karpurapu, Z. Liu, C. Zhang, N. Dronadula, and G. N. Rao
Suppression of Activation of Signal Transducer and Activator of Transcription-5B Signaling in the Vessel Wall Reduces Balloon Injury-Induced Neointima Formation
Am. J. Pathol., October 1, 2007; 171(4): 1381 - 1394.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
D. Wang, Z. Liu, Q. Li, M. Karpurapu, V. Kundumani-Sridharan, H. Cao, N. Dronadula, F. Rizvi, A. K. Bajpai, C. Zhang, et al.
An Essential Role for gp130 in Neointima Formation Following Arterial Injury
Circ. Res., March 30, 2007; 100(6): 807 - 816.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K.-S. Kim, S. Sengupta, M. Berk, Y.-G. Kwak, P. F. Escobar, J. Belinson, S. C. Mok, and Y. Xu
Hypoxia Enhances Lysophosphatidic Acid Responsiveness in Ovarian Cancer Cells and Lysophosphatidic Acid Induces Ovarian Tumor Metastasis In vivo
Cancer Res., August 15, 2006; 66(16): 7983 - 7990.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. Cao, N. Dronadula, F. Rizvi, Q. Li, K. Srivastava, W. T. Gerthoffer, and G. N. Rao
Novel Role for STAT-5B in the Regulation of Hsp27-FGF-2 Axis Facilitating Thrombin-Induced Vascular Smooth Muscle Cell Growth and Motility
Circ. Res., April 14, 2006; 98(7): 913 - 922.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
H. Cao, N. Dronadula, and G. N. Rao
Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells
Am J Physiol Cell Physiol, January 1, 2006; 290(1): C172 - C182.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/4/3112    most recent
M409739200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dronadula, N.
Right arrow Articles by Rao, G. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dronadula, N.
Right arrow Articles by Rao, G. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement