| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Physiology and Vascular Biology Center, University of Tennessee, Memphis, Tennessee 38163
Submitted 30 December 2002 ; accepted in final form 21 April 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
phosphatase activity; protein tyrosine phosphorylation; overexpression; Cas-Crk association
That nitric oxide (NO) decreases the motility of subcultured aortic smooth muscle cells from the adult rat and primary aortic smooth muscle cell cultures from the newborn rat is now well established (28, 40). Moreover, vascular smooth muscle cell proliferation, motility, and neointimal development are attenuated by overexpression of endothelial cell NO synthase in the rat carotid artery (48), whereas inhibition of NOS increases neointima formation (33). These findings are consistent with the view that NO has the capacity to decrease cell motility in vivo.
Protein tyrosine phosphorylation, a reversible and dynamic process, is considered to be an important component of the mechanism regulating cell motility (19). The phosphorylation levels of tyrosine-containing proteins are regulated by the opposite actions of two enzyme families, that is, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Several kinases have been identified in regulation of vascular smooth muscle cell motility (21, 34, 35). Conversely, recent reports (28, 44) have indicated a role of PTP-1B in inhibition of vascular smooth muscle cell motility induced by NO.
Focal adhesions are subcellular domains linking integrins to the actin cytoskeleton. These structures are considered essential elements for transduction of cell movement and are major sites of dynamic tyrosine phosphorylation and dephosphorylation in cells (1, 9). Integrin binding to extracellular matrix proteins is thought to trigger auto/transphosphorylation and activation of a cytoplasmic PTK, focal adhesion kinase (FAK). FAK is then thought to interact with Src kinase, inducing tyrosine phosphorylation of several focal adhesion adapter proteins, including p130cas (Crkassociated substrate), paxillin, or Shc (26, 41). Both paxillin and p130cas are also substrates for several PTPs, including PTP-1B and PTP-PEST (32, 42, 43, 46). Integrin-induced phosphorylation of p130cas promotes the association of p130cas with another adapter protein, Crk (for chicken tumor virus No. 10 regulator of kinase) (20). Cas-Crk association is thought to be a "molecular switch" for induction of cell motility (30).
PTP-PEST is an important regulator of cell motility, as shown by decreased motility in fibroblasts overexpressing wild-type PTP-PEST (25). On the other hand, knockout of PTP-PEST in mouse embryonic fibroblasts also induces an increase in cell spreading and a strong defect in cell motility on fibronectin (1). An explanation for these contradictory results is that an intermediate level of PTP-PEST may be required to maintain cell motility. PTP-PEST has been reported to recognize the aforementioned focal adhesion adapter proteins as well as the PTK FAK as substrates (1).
Primary cultures of aortic smooth muscle cells isolated from newborn rats express a partially dedifferentiated cytoskeletal phenotype resembling that of neointimal smooth muscle cells (3). Nevertheless, these cells maintain high levels of protein kinase G (PKG) activity relative to subcultured cells, which are known to have low levels of PKG (unpublished observations). Because PKG levels also remain high in neointimal cells (6), cultured cells from newborn rats represent an excellent in vitro model to investigate mechanisms related to vascular smooth muscle cell motility. These findings prompted us to test the hypothesis that NO inhibits the motility of rat aortic smooth muscle cells via a mechanism involving dephosphorylation of focal adhesion proteins by PTP-PEST. We report the novel findings that NO upregulates PTP-PEST activity in aortic smooth muscle cells and that this event is both necessary and sufficient to explain the NO-induced inhibition of cell motility. We also report that dephosphorylation of p130cas, but not paxillin or FAK by PTP-PEST, is a downstream event that is both necessary and sufficient to mediate NO-induced antimotogenesis. Finally, we show that PTP-PEST mediates the capacity of NO to induce dissociation of p130cas and the protooncogene Crk.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
|
|
|
Cell culture. Smooth muscle cells were obtained from the thoracic
aortas of newborn Sprague-Dawley rats (6–9 days old), as previously
described (16). This study was
performed via a protocol approved by the Animal Care and Use Committee of the
University of Tennessee Health Science Center (Memphis, Tennessee) in
accordance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals (NIH Pub. No 86-23). The cells were seeded into
Primaria culture plates at a density of 1.8–2.3 x 104
cells/cm2. They were then cultured for the first 2 days in
serum-free DMEM/Ham's F-12 (1:1) medium, supplemented with insulin (5
µg/ml), transferrin (5 µg ml), and selenous acid (5 ng/ml) plus 50 U/ml
penicillin and 50 µg/ml streptomycin in a humidified atmosphere of 5%
CO2-95% air. Most cells (
95%) attached to the culture surface
within the first few hours after being seeded and were observed to spread
after overnight incubation. After the initial 2-day culture in serum-free
medium, fetal bovine serum was added to a final concentration of 10%, and
culture was continued in DMEM/F-12 lacking insulin-transferrin-selenous acid.
Cells were cultured for an additional 3–5 days in 10% serum-containing
medium, whereupon they reached confluence. Cells were then cultured in
serum-free medium for 2 days to induce motogenic and mitogenic quiescence.
Most experiments in this study were performed using quiescent cells in primary
culture. Selected experiments utilized subcultured cells of up to passage
2, as indicated.
Preparation of recombinant adenovirus expressing wild-type or mutant PTP-PEST or p130cas. Replication-deficient (E1 deleted) recombinant type 5 adenovirions expressing wild-type or mutant PTP-PEST were prepared via the use of a commercial kit (Adeno-X) purchased from Clontech. Plasmid (pGEM3Z-PTPPEST) containing mouse sequence PTP-PEST was generously provided by Dr. Andre Veillette (Montreal, Quebec, Canada). This cDNA was sequenced, and any variances from the published sequence were corrected by site-directed mutagenesis (12). A Kozak sequence and a 9-amino acid hemagglutinin epitope at the NH2-terminus, as well as flanking XbaI and BstXI restriction sites, were introduced by PCR. The PCR product was subcloned into the Adeno-X-pShuttle vector via XbaI/BstXI restriction sites. The mutation to generate catalytically inactive C231S-PTP-PEST was introduced via the use of a QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Wild-type PTP-PEST and C231S-PTP-PEST cDNAs in the pShuttle vector were sequenced and conformed to published data (12). An I-CeuI/PI-SceI restriction fragment from pShuttle containing the cytomegalovirus IE promoter/enhancer 5' to the cDNA insert and the bovine growth hormone polyadenylation signal 3' to the insert was ligated into adenoviral DNA backbone, also restricted with I-CeuI and PI-SceI. After amplification and purification of recombinant viral DNA from bacteria, adenovirus was produced by transfecting Pac 1-linearized recombinant viral DNA into HEK-293 cells via the use of the lipid transfection agent Fugene 6.
Adenoviruses expressing wild-type or mutant p130cas were
generated via a kit generously provided by Dr. Beverly Davidson (University of
Iowa, Iowa City, IA). Plasmid pSSR
, containing the wild-type rat cDNA
of p130cas and Cas mutant cDNA with an in-frame deletion of its
substrate domain (CAS-SD, amino acids 213–514), was kindly provided by
Dr. Kristiina Vuori (La Jolla, California). A ClaI restriction site
and Kozak sequence at the 5' end and a NotI and hemagglutinin
tag sequence at the 3' end of p130cas cDNA were introduced by
PCR. Wild-type and mutant p130cas were subcloned into the pShuttle
vector (pAd5CMV k-NpA, No. 968, Gene transfer vector core, University of Iowa)
via the ClaI/NotI-restricted pShuttle vector
(39). Recombinant adenovirus
was obtained by cotransfection of HEK-293 cells with the pShuttle vector DNA
containing insert p130cas gene and adenoviral backbone DNA (pAd5
9.2-100, No. 975, gene transfer vector core, University of Iowa).
Adenoviral titers were determined via a standard procedure by measurement of their cytopathic effect in HEK-293 cells.
Infection of cells with recombinant adenovirus and transfection of
cells with plasmid DNA. Confluent cells were infected with recombinant
adenovirus at a multiplicity of infection index of 5–10. After the
initial 24-h incubation, the medium was replaced, and cells subjected to a
second 24-h incubation period before use in experiments. Confluent cells were
also transfected with the plasmid pCAGG-Crk II, which expressed myc-tagged
wild-type Crk II under the control of a 
-actin promoter. This plasmid
was generously donated by Dr. Kristiina Vuori. For transfection experiments,
cells were incubated with or without 3 µg of plasmid DNA pUC19 or wild-type
Crk II plasmid DNA pCAGG-Crk II using Fugene 6 as a transfection reagent,
according to the manufacturer's instructions. Media were replaced, and cells
were incubated for a second 24 h before measurement of cell motility.
Measurement of PTP-PEST activity via immunophosphatase assay. After treatment with the NO donors SNAP or DETA/NO or the control substance NAP, cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 1% Triton X-100, and 50 mM Tris; pH 7.2) containing protease inhibitor cocktail (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 µM aprotinin, 20 µM leupeptin, 40 µM bestatin, 15 µM pepstatin A, and 14 µM E-64). Immunoprecipitation was carried out by preclearing lysates with normal rabbit serum for 30 min at 4°C and further with 50 µl of protein G-Sepharose beads (50%) for 30 min. The supernatant was incubated with 5 µlof PTP-PEST antiserum overnight at 4°C and further with 50 µl of protein G-Sepharose beads for 2 h. Immunocomplexes were washed twice with the above-mentioned RIPA buffer and twice with ice-cold HEPES-DTT buffer [25 mM HEPES (pH 7.3), 5 mM EDTA, and 10 mM DTT]. Immunoprecipitated PTP-PEST beads were incubated with 32(P)poly(Glu, Tyr) (100,000 cpm) for 30 min (a time point that is within the linear portion of the time-vs.-activity relationship, data not shown). 32P-poly(Glu, Tyr) was phosphorylated by Src kinase via a previously published procedure (18). The phosphatase reaction was stopped by the addition of 2 mg/ml BSA and 20% TCA, which precipitated unhydrolyzed substrate, leaving inorganic phosphate in the supernatant, which was then measured by scintillation spectrometry. Incubation of 32P-poly(Glu, Tyr) without immunoprecipitated PTP-PEST was used to determine background values. Immunoprecipitation with preimmune serum was used as a nonspecific control. The activity of nonspecifically precipitated PTP was <22% of the activity of immunoprecipitated PTP-PEST. For the immunophosphatase assay using pNPP, immunoprecipitation of PTP-PEST was carried out essentially as described above. Immunoprecipitated PTP-PEST was then incubated with 10 mM pNPP for 1 h at 37°C (a time point that is within the linear portion of the time-vs.-activity relationship, data not shown). The enzyme activity was determined by measuring the increase of absorbance at 405 nm. The values in the absence of the enzyme were taken as background and were always subtracted. The reaction was terminated by the addition of 10 M NaOH.
Immunoprecipitation and immunoblotting. Cells were lysed in RIPA
buffer (150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 50
mM Tris; pH 7.2) containing 1 mM sodium vanadate, 1 mM PMSF, 5 µg/ml
aprotinin, and 5 µg/ml leupeptin. Protein concentration in lysates was
determined by the bicinchoninic acid method. Immunoprecipitation was done by
incubation of cell lysates (0.5 mg protein) with antibodies directed
against p130cas, paxillin, or FAK overnight at 4°C, followed by
the addition of protein G-Sepharose beads (Pharmacia) and further incubation
for 2 h at 4°C. Sepharose beads were then washed twice with the
above-mentioned buffer and once with 50 mM Tris buffer (pH 7.2) containing 150
mM NaCl, 1 mM sodium vanadate, and 1 mM PMSF, followed by boiling of beads in
2x Laemmli sample buffer and loading of supernatant onto gels for
SDS-PAGE. Proteins were transferred electrophoretically onto polyvinylidene
difluoride (PVDF) membranes (Immobilon PVDF, Millipore), and Ponceau S (0.5%,
Sigma) was used to confirm transfer of equivalent amounts of protein. In some
cases, blots were reprobed with anti--actin antibody to confirm
equivalent loading of proteins. After the transfer, blots were probed with
primary antibodies against PTP-PEST, p130cas, paxillin, or FAK,
followed by horseradish peroxidase-coupled secondary antibodies with
appropriate specificity. The blots were then stripped and reprobed with
anti-phosphotyrosine antibodies (1:10,000). Immunoreactive bands were
visualized using Renaissance chemiluminescence reagents (DuPont/NEN). Some
blots were first probed with anti-phosphotyrosine antibody (horseradish
peroxidase conjugate). The blots were then stripped and probed with
anti-p130cas antibody. Band densities were measured using NIH Image
software.
Measurement of cell migration. Confluent cells in a six-well plate were incubated with serum-free medium with or without recombinant adenovirus for 24 h, and the medium was then replaced by fresh serum-free medium. The cells were incubated for another 24 h in this medium to allow time for transgenic protein expression. A 0.25-mm-wide scratch was then made on the bottom of the plate with a sterile 200-µl pipette tip. The cells were washed with serum-free medium once. Images of the wounded area were collected digitally using a charge-coupled device camera and AppleVideo software. The cells were incubated in DMEM/Ham's F-12 medium containing 0.05% serum and 5 mM hydroxyurea with or without 100 µM SNAP for 24 h. Hydroxyurea was used to prevent cell proliferation as previously described (6). Images of the same site were collected again as described above and were analyzed via NIH Image software.
Statistical analysis. Data are expressed as means ± SE and were statistically evaluated using ANOVA, followed by Fisher's protected least-significant-difference test or randomized complete block ANOVA for the raw data, or via paired, two-tailed Student's t-test. P < 0.05 was considered to be significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Treatment of cells with two NO donors, SNAP and DETA/NO, increased PTP-PEST activity in concentration-dependent fashion (Fig. 1, A and C). Both SNAP (100 µM) and DETA/NO (100 µM) increased the activity of PTP-PEST by about threefold. On the other hand, NAP (100 µM), an inactive analog of SNAP that lacks the NO moiety, had no significant effect (Fig. 1B), indicating that the effect of SNAP was attributable to its capacity to serve as a NO donor. Sodium orthovana-date (10 mM), a general inhibitor of phosphatase activity, abolished PTP-PEST activity (data not shown). In addition, immunoprecipitation with preimmune serum was used as nonspecific control in this assay. We found that the activity of nonspecifically precipitated PTP-PEST accounted for <22% of the activity of immunoprecipitated PTP-PEST (data not shown). Moreover, we verified that SNAP (100 µM) did not significantly affect PTP-PEST activity when it was directly incubated with immunoprecipitated PTP-PEST for 30 min (data not shown). Figure 1D shows the relationship between the time of incubation of cells with SNAP and the activity of PTP-PEST. As shown, activity remained significantly elevated for at least 24 h.
|
Increased PTP-PEST activity is potentially attributable to increased PTP-PEST protein levels. To test whether NO enhanced the protein levels of PTP-PEST, cells were treated with 100 µM SNAP for periods ranging from 30 min to 24 h. PTP-PEST levels were then measured by Western blot analysis. As shown in Fig. 2, SNAP failed to increase PTP-PEST protein levels, indicating that NO induced an increase of specific PTP-PEST enzyme activity rather than increasing enzyme protein levels.
|
cGMP analog increases the activity of PTP-PEST, whereas guanylyl cyclase inhibitor blocks activation of PTP-PEST induced by NO donor. It is well accepted that NO increases cGMP levels by activating cytosolic guanylyl cyclase (7). To test the hypothesis that NO activates PTP-PEST via the cGMP pathway, we performed two types of experiments. First, cells were treated with 100 µM 8-pCPT-cGMP, an analog of cGMP. PTP-PEST activity was then measured via immunophosphatase assay. As shown in Fig. 3A, treatment of cells with 8-pCPT-cGMP induced a significant increase of PTP-PEST activity. Second, cells were pretreated with a selective guanylyl cyclase inhibitor, ODQ, for 30 min before treatment with the NO donor SNAP. As shown in Fig. 3B, ODQ blocked the capacity of SNAP to increase PTP-PEST activity. These data indicate that elevation of cGMP is not only sufficient, but also necessary, for NO-induced PTP-PEST activation.
|
Overexpression of wild-type PTP-PEST mimics, whereas overexpression of dominant negative PTP-PEST reverses, the inhibitory effect of NO on cell motility. In view of the increased PTP-PEST activity in response to NO, we were prompted to test the hypothesis that this effect is sufficient to explain NO-induced inhibition of cell motility. Thus primary aortic smooth muscle cells were infected with recombinant adenovirus expressing wild-type PTP-PEST, and cell motility was then measured. As shown in Fig. 4, overexpression of wild-type PTP-PEST mimicked the inhibitory effect of NO on cell motility, indicating that upregulation of PTP-PEST is sufficient to explain the antimotogenic effect of NO. To test the hypothesis that PTP-PEST is necessary for NO-induced inhibition of motility, we treated cells with adenovirus expressing C231S-PTP-PEST, an allele that is catalytically inactive. The results shown in Fig. 4 indicate that expression of C231SPTP-PEST blocked the capacity of NO to decrease cell motility. These results, together with the above data, indicate that PTP-PEST is both sufficient and necessary to explain the NO-induced effects on cell motility.
NO donor and PTP-PEST induce reduction of p130cas phosphotyrosine levels, whereas dominant negative PTP-PEST blocks the decrease of p130cas phosphotyrosine levels induced by NO. On the basis of previous findings indicating an association between increased phosphotyrosine levels of p130cas and cell motility (25, 31), we tested the hypothesis that a NO donor would reduce focal adhesion protein phosphotyrosine levels via a mechanism requiring upregulation of PTP-PEST. As shown in Fig. 5, the NO donor decreased p130cas phosphotyrosine levels. Moreover, overexpression of wild-type PTP-PEST induced a similar effect, indicating that upregulation of PTP-PEST is sufficient to explain the capacity of NO to decrease p130cas phosphorylation levels. Conversely, expression of dominant negative C231S-PTP-PEST blocked the capacity of NO to reduce p130cas phosphotyrosine levels. These results indicate that PTP-PEST is both necessary and sufficient to explain the capacity of NO to modulate phosphotyrosine levels in p130cas. The data also suggest that decreased cell motility induced by NO could be explained via reduction of p130cas phosphotyrosine levels in aortic smooth muscle cells.
Overexpression of wild-type PTP-PEST fails to decrease phosphotyrosine levels in the focal adhesion proteins paxillin or FAK. Phosphotyrosine levels of both paxillin and FAK are reduced in aortic smooth muscle cells by treatment with a NO donor (28). Moreover, both proteins appear to play a key role in regulation of cell motility (8, 45), and they have been reported to serve as substrates for PTP-PEST, although the latter aspect is controversial (1, 25, 43). To test the hypothesis that paxillin and FAK function in vivo as direct or indirect substrates for PTP-PEST, we overexpressed wild-type PTP-PEST and measured paxillin and PTP-PEST phosphotyrosine levels via immunoprecipitation, followed by Western blotting. As shown in Fig. 6, neither paxillin nor FAK phosphotyrosine levels were reduced in PTP-PEST-overexpressing cells, despite concomitant dephosphorylation of p130cas in the same experiments.
Substrate domain-deleted p130cas mutant mimics, whereas wild-type p130cas blocks, the effect of NO on cell motility. To establish the functional relevance of p130cas dephosphorylation induced by NO or PTP-PEST, we determined the effect of expression of a mutant p130cas allele (SD-Cas) lacking the substrate domain that contains multiple tyrosine phosphorylation sites (30). We reasoned that if dephosphorylation of substrate domain phosphotyrosine was functionally important for inhibition of cell motility, expression of a mutant lacking this domain should mimic the capacity of NO or PTP-PEST to induce inhibition of cell motility. Indeed, as shown in Fig. 7, expression of SD-Cas markedly decreased cell motility. Conversely, we anticipated that overexpression of wild-type p130cas would increase cell motility and antagonize the motility inhibitory effect of NO (30). In accordance with this expectation, the data shown in Fig. 7 indicate that overexpression of wild-type p130cas increased motility and markedly attenuated the capacity of NO to decrease motility. Separately, we found that overexpression of wild-type p130cas was associated with increased phosphotyrosine levels in p130cas (data not shown). The latter finding is consistent with data obtained by other investigators (30) and supports the view that overexpressed p130cas induces increased cell motility via a mechanism dependent on p130cas tyrosyl phosphorylation. Taken together, these findings support the hypothesis that tyrosine dephosphorylation of p130cas is both necessary and sufficient to explain the NO-induced inhibition of cell motility.
|
NO decreases association of p130cas with protooncogene Crk, and overexpression of wild-type PTP-PEST mimics, whereas overexpression of dominant negative PTP-PEST blocks, NO-induced Cas-Crk dissociation. Association of the protooncogene Crk with p130cas has been found to increase upon phosphorylation of p130cas after integrin stimulation (20). Moreover, Cas-Crk association appears to be essential for induction of cell motility (30). To identify events downstream of p130cas in NO-induced signal transduction pathways, we tested the hypothesis that a NO donor would reduce association of p130cas with Crk, via a mechanism requiring upregulation of PTP-PEST. As shown in Fig. 8, the NO donor decreased Cas-Crk coimmunoprecipitation. Moreover, overexpression of wild-type PTP-PEST induced a similar effect, indicating that upregulation of PTP-PEST is sufficient to explain the inhibitory effect of NO. Conversely, expression of dominant negative C231S-PTP-PEST blocked the capacity of NO to decrease Cas-Crk association. These results indicate that PTP-PEST is both necessary and sufficient to explain the capacity of NO to induce dissociation of Cas-Crk complexes. The data also suggest that decreased cell motility induced by NO could be explained via a reduction of Cas-Crk association.
p130cas coimmunoprecipitates with the substrate-trapping PTP-PEST allele C231S-PTP-PEST. The aforementioned results suggest that p130cas can function as a substrate of PTP-PEST. To investigate whether PTP-PEST directly interacts with p130cas in rat aortic smooth muscle cells, we infected cells with control adenovirus expressing enhanced green fluorescent protein, adenovirus expressing wild-type PTP-PEST, or adenovirus expressing C231S-PTP-PEST. The latter protein has been shown to function as a substrate-trapping protein by other investigators (14, 22, 23). Thus we immunoprecipitated PTP-PEST and measured the levels of coimmunoprecipitated p130cas via Western blot analysis. The quantity of immunoprecipitated PTP-PEST was measured by reprobing the same blot with anti-PTP-PEST. As shown in Fig. 9, overexpression of C231S-PTP-PEST increased the association of p130cas with PTP-PEST significantly. Immunoprecipitation of wild-type PTP-PEST failed to coimmunoprecitate p130cas, in agreement with previous reports (14, 23). These data are consistent with the view that p130cas is a substrate for PTP-PEST in rat aortic smooth muscle cells. The data are also consistent with the finding that overexpression of wild-type PTP-PEST decreases phosphotyrosine levels in p130cas.
|
Overexpression of wild-type Crk II rescues the inhibitory effect of wild-type PTP-PEST. The aforementioned results indicate that Crk is a pivotal downstream signal transduction element in aortic smooth muscle cell motility. If Crk were indeed operating downstream of PTP-PEST, then overexpression of Crk should rescue the inhibitory effect of PTP-PEST. Indeed, as shown in Fig. 10, the inhibitory effect of PTP-PEST was blocked by simultaneous overexpression of Crk, indicating its downstream involvement in the signal transduction pathways influenced by NO and PTP-PEST.
|
Crk increases cell motility both in the presence and absence of dominant negative PTP-PEST. It is unlikely that the C231S mutant PTP-PEST would have an effect other than as dominant negative, because it did not have an effect of its own on motility (Figs. 4 and 10). However, to obtain additional data regarding this aspect, we determined whether the effect of a downstream motility activator (in this case, Crk) would be altered by expression of dominant negative PTP-PEST. We reasoned that if dominant negative PTP-PEST were to have a global effect on motility, it would likely interfere with downstream signal transduction pathways involving cell motility. As shown in Fig. 10, overexpression of Crk II induced equivalent increases of cell motility, both in the presence or absence of dominant negative PTP-PEST, further supporting the notion that the effect of C231S-PTP-PEST against NO is both upstream of Crk and not the result of a nonspecific effect.
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
We (5, 11) have found that the motility stimulatory effect of NO in differentiated cells is linked to upregulation of PTP Src homology 2 phosphatase 2 (SHP2) via a cGMP-mediated mechanism. We (44) have also found that NO increases the activity of PTP-1B, a nonreceptor PTP, in less differentiated rat aortic smooth muscle cells isolated from newborn rats and that this event is both necessary and sufficient to explain the NO-induced inhibition of cell motility.
In the current study, we targeted the role of a related PTP, PTP-PEST, in NO-induced antimotogenesis. PTP-PEST is a ubiquitously expressed phosphatase; moreover, a recent study (49) has reported that the rat carotid artery expresses significant levels of mRNA for PTP-PEST. PTP-PEST recognizes many of the substrates targeted by PTP-1B, including several focal adhesion proteins such as p130cas, paxillin, and FAK. PTP-PEST could therefore potentially play a role in transduction of NO-induced antimotogenesis. However, the substrates for PTP-PEST in vascular smooth muscle cells have not yet been completely identified.
In the present study, we report the novel findings that NO increases the enzyme activity of PTP-PEST and that this event is both necessary and sufficient to account for the NO-induced inhibition of cell motility in primary cultures of relatively dedifferentiated aortic smooth muscle cells from newborn rats. Moreover, we found that the inhibitory effect of NO requires cGMP as a second messenger. We also provide evidence that PTP-PEST-induced phosphotyrosine dephosphorylation of the adapter protein p130cas induces dissociation of p130cas from the adapter protein Crk and that this is an obligatory downstream event involved in NO-induced inhibition of cell motility.
Our results support the hypothesis that the antimotogenic effect of NO requires upregulation of PTP-PEST and that PTP-PEST activity is essential, as demonstrated by the dominant negative effect of catalytically inactive PTP-PEST expression.
The precise mechanism for activation of PTP-PEST by NO is not known. We
found that the addition of the NO donor DETA/NO after immunoprecipitation did
not significantly change the activity of PTP-PEST, suggesting that NO may not
directly regulate the activity of PTP-PEST (unpublished data). We also did not
find a significant change of PTP-PEST activity when the latter was incubated
directly with 8-pCPT-cGMP (unpublished data). Exposure of PTP-PEST by
PKG-I in vitro also failed to alter the activity of PTP-PEST
(unpublished data). A previous study
(29) has shown that NO and
other cGMP agonists induce dephosphorylation of focal adhesion proteins via a
reduction of intracellular calcium. Other groups
(18,
37) have reported that an
increase in intracellular calcium is associated with a reduction of PTP
activity. We found that the stimulatory effect of NO on PTP-PEST requires cGMP
as a second messenger. Data from other laboratories indicate that PKC inhibits
PTP-PEST activity by phosphorylating serine residue 39
(24). Therefore, another
mechanism such as tyrosine and/or serine dephosphorylation or phosphorylation
of PTP-PEST regulated by the cGMP-dependent pathway may be the major factor
for activation of PTP-PEST in our experiments. However, these possibilities
remain the subject of future experiments.
The capacity of overexpressed PTP-PEST to decrease cell motility in aortic smooth muscle cells is similar to that reported in some but not all studies. Thus mouse fibroblasts that overexpress PTP-PEST manifest a defect in cell motility that coincides with significantly reduced levels of phosphotyrosine levels in p130cas (25). Paradoxically, however, fibroblasts from PTP-PEST-null embryos also migrate more slowly (1). While the reason(s) for this discrepancy are not clear, other investigators (15) have speculated that an intermediate level of expression may be necessary for optimal cell motility.
Our experiments utilizing overexpression of wild-type and substrate domain-deleted p130cas indicate that the biochemical mechanism underlying the antimotogenic capacity of PTP-PEST is obligatorily linked to dephosphorylation of p130cas. This is consistent with a previous study (14) indicating that PTP-PEST recognizes p130cas as a substrate, via the NH2-terminal proline-rich region of PTP-PEST and the SH3 domain of p130cas. Our data further indicate that NO induces dissociation of p130cas from Crk, through upregulation of PTP-PEST. Cas-Crk complexes have been shown to control actin dynamics via activation of Rac1, one member of the Rho family of small GTPases (30). DOCK180, a prominent 180-kDa Crk SH3 domain binding protein, has been shown to link Cas-Crk complexes to Rac1 activation (4). It has been shown that Rac is essential in the formation of protrusion of lamellipodia and pseudopodia at the leading edge of the cells, resulting in cell movement (13, 47). Furthermore, Rac may play a role in cell retraction at the trailing edge (47).
Several, but not all, studies have reported that PTP-PEST can also induce dephosphorylation of the adapter protein paxillin. Although we (28) have previously reported that NO induces paxillin dephosphorylation, overexpression of PTP-PEST in the present study failed to elicit this effect, indicating that paxillin does not function as a substrate for PTP-PEST in the aortic smooth muscle cells used in the current study. Moreover, we speculate that the phosphatase involved in dephosphorylating paxillin may be PTP-1B, inasmuch as NO also increases PTP-1B activity (28), but this aspect remains to be investigated. Other studies have indicated that FAK may also be a substrate for PTP-PEST, but, similar to paxillin, we were unable to observe decreased FAK phosphorylation in PTP-PEST-overexpressing cells.
Migration of vascular smooth muscle cells from the media to the intima and proliferation of vascular smooth muscle cells in the intima are key events in the early stages of atherosclerosis and restenosis after vascular injury (38). The present data may therefore have potential pathophysiological significance. The motility of vascular smooth muscle cells is regulated by cell-matrix interaction and growth factors, which are released during vascular injury (36). The levels of inducible NO synthase are strongly and rapidly increased after experimental vascular injury (2, 27), and the induction of inducible NO synthase has been shown to increase the levels of NO (27). Our results indicate that NO attenuates cell motility by dephosphorylation of p130cas and disassembly of Cas-Crk complexes via upregulation of PTP-PEST. Therefore, it is likely that NO may play a counterregulatory role in modulating vascular smooth muscle cell motility in vascular injury. However, extrapolation of our results to in vivo mechanisms should be done with caution inasmuch as the formation of neointima in vascular injury is subject to multiple simultaneous influences.
In summary, we have shown that upregulation of PTP-PEST can explain the capacity of NO to reduce cell motility, dephosphorylation of p130cas, and disassociation of Cas-Crk complexes.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Current address of Y. Lin: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. D. Tang p130 Crk-Associated Substrate (CAS) in Vascular Smooth Muscle Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2009; 14(2): 89 - 98. [Abstract] [PDF]
|
|
|
|
 |
|
 L. P. Desai, S. E. Sinclair, K. E. Chapman, A. Hassid, and C. M. Waters High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L769 - L778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Halle, Y.-C. Liu, S. Hardy, J.-F. Theberge, C. Blanchetot, A. Bourdeau, T.-C. Meng, and M. L. Tremblay Caspase-3 Regulates Catalytic Activity and Scaffolding Functions of the Protein Tyrosine Phosphatase PEST, a Novel Modulator of the Apoptotic Response Mol. Cell. Biol., February 1, 2007; 27(3): 1172 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-C. Ceacareanu, B. Ceacareanu, D. Zhuang, Y. Chang, R. M. Ray, L. Desai, K. E. Chapman, C. M. Waters, and A. Hassid Nitric oxide attenuates IGF-I-induced aortic smooth muscle cell motility by decreasing Rac1 activity: essential role of PTP-PEST and p130cas Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1263 - C1270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chang, B. Ceacareanu, D. Zhuang, C. Zhang, Q. Pu, A. C. Ceacareanu, and A. Hassid Counter-Regulatory Function of Protein Tyrosine Phosphatase 1B in Platelet-Derived Growth Factor- or Fibroblast Growth Factor-Induced Motility and Proliferation of Cultured Smooth Muscle Cells and in Neointima Formation Arterioscler Thromb Vasc Biol, March 1, 2006; 26(3): 501 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Giordano, D. M. Magaletti, and E. A. Clark Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19 Blood, February 15, 2006; 107(4): 1537 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. I. Palen, S. Belmadani, P. A. Lucchesi, and K. Matrougui Role of SHP-1, Kv.1.2, and cGMP in nitric oxide-induced ERK1/2 MAP kinase dephosphorylation in rat vascular smooth muscle cells Cardiovasc Res, November 1, 2005; 68(2): 268 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zhuang, A.-C. Ceacareanu, B. Ceacareanu, and A. Hassid Essential role of protein kinase G and decreased cytoplasmic Ca2+ levels in NO-induced inhibition of rat aortic smooth muscle cell motility Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1859 - H1866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kappert, K. G. Peters, F. D. Bohmer, and A. Ostman Tyrosine phosphatases in vessel wall signaling Cardiovasc Res, February 15, 2005; 65(3): 587 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chang, D. Zhuang, C. Zhang, and A. Hassid Increase of PTP levels in vascular injury and in cultured aortic smooth muscle cells treated with specific growth factors Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2201 - H2208. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
