| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Cancer Centrum Karolinska, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden;
Division of Vascular Surgery, Uppsala University Hospital, Uppsala, Sweden; and
Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden
1 Correspondence: Cancer Centrum Karolinska, Department of Oncology-Pathology, Karolinska Institutet, 17176 Stockholm, Sweden. E-mail: arne.ostman{at}ki.se
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ß receptors, and five PTPs implied in control of PDGF-receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF-ß receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression—particularly, DEP-1 levels—appeared to be the dominating factor determining receptor-phosphorylation and VSMC proliferation. In support of these findings, cultured DEP-1–/– cells displayed increased PDGF-dependent cell signaling. Hyperactivation of PDGF-induced signaling was also observed after siRNA-down-regulation of DEP-1 in VSMCs. The results indicate a previously unrecognized role of PDGF-receptor-targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK-dependent tissue remodeling.
Key Words: tissue remodeling • laser capture microdissection • vascular smooth muscle cell • protein tyrosine phosphatase
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). RTKs are involved in numerous developmental processes involving physiological tissue remodeling during development but also contribute to pathological processes, such as restenosis and different conditions associated with fibrosis. The vast majority of studies on these aspects of RTK action have focused on the transcriptional up-regulation of RTKs, or their cognate ligands, as the major determinants of onset and termination of these processes.
The PDGF- and PDGF-ß tyrosine kinase receptors, and their ligands, are potent regulators of mesenchymal cells such as fibroblasts, pericytes and smooth muscle cells. The human genome encodes four different PDGF isoforms, PDGF-A, -B, -C, and -D. The different PDGF chains assemble into heterodimers and homodimers: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. These differ in their receptor-binding such that PDGF-AA, -AB, -BB, and -CC induce receptor homodimers, PDGF-AB and -BB induce ß receptor heterodimers, and finally PDGF-BB and -DD lead to ßß receptor homodimers (reviewed in (2)
).
Like other RTKs, the PDGF receptors are negatively regulated by protein tyrosine phosphatases (PTPs) (3
, 4)
. PTPs constitute a diverse family of receptor-like and cytosolic proteins (5)
. Individual PTPs display high substrate specificity, exemplified by pathway-specific alterations in signaling via the receptors of PDGF, insulin-like growth factor (IGF)-1, and insulin on depletion of different PTPs (6
7
8)
. Recent studies have implied DEP-1, PTP-1B, TC-PTP, and PTP as negative regulators of PDGF receptor signaling (6
, 9
, 10)
. Various mechanisms for control of the specific activity of PTPs have been described, including phosphorylation, reversible oxidation of the active-site cysteine, and in the case of receptor-like PTPs, binding of extracellular ligands. In addition to this, transcriptional regulation is another major mechanism controlling the activity of PTPs (11
12
13)
.
PDGF receptor overactivity has been implicated in a number of fibrotic and chronic inflammatory diseases, such as liver fibrosis, glomerulonephritis, and atherosclerosis (14
, 15)
. Restenosis after angioplasty-associated injury of the vessel wall represents an additional PDGF-dependent pathological process, which involves tissue remodeling (16)
. Restenosis is characterized by the proliferation and migration of vascular smooth muscle cells (VSMCs), and the subsequent deposition of collagens and other extracellular matrix (ECM) proteins, which finally leads to neointima formation and vascular narrowing. A series of studies with different types of PDGF antagonists have established a causal role of PDGF receptor signaling in this process. Concerning the different PDGF ligands and their role in restenosis, the importance of PDGF-A and PDGF-B in vascular diseases is well documented (reviewed in (15)
). Recently, also PDGF-D has attracted some attention due to their mitogenic effects on VSMCs and stimulatory effects on neointima formation (17
, 18)
.
In contrast, the functional role of PTPs in controlling restenosis has not been well studied. Some preliminary observations support the notion of a regulatory role of PTPs in restenosis. These include the findings of growth factor-dependent modulation of PTP expression in VSMCs in vitro, as well as counteractivity of PTP1B on PDGF-dependent VSMC motility (19
, 20)
.
In this study, we have therefore used PDGF-dependent injury-induced neointima formation, as a prototypic process of RTK-regulated tissue remodeling, to analyze the contribution of different components—ligands, receptors, receptor-antagonizing PTPs—in determining onset and termination of the process. This has been done by vessel wall layer-specific analyses of the expression of PDGF ligands, receptors, and phosphatases and by correlation of these results with the spatial and temporal pattern of PDGF-ß receptor phosphorylation and cell proliferation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), surgically extracted, and snap frozen together with the corresponding noninjured artery of the contralateral side 8 days and 14 days post intervention (n=5 per group). Three hours before artery resection, the animals received an intravenous (i.v.) injection of 7 mg 5-bromo2'-deoxy-uridine (bromodeoxyuridine (BrdU), Sigma, Stockholm, Sweden) diluted in 40% ethanol. The experimental protocol was approved by the Uppsala University Ethics Committee according to the European Union guidelines.
Laser capture microdissection and real-time polymerase chain reaction
The media and the neointima of the injured arteries (day 8 and day 14) as well as the media of the control arteries (day 8 and day 14) were microdissected separately from frozen tissue sections (n=8 per animal), using the Arcturus laser microscope (Arcturus Bioscience, Mountain View, USA). RNA was isolated with the Arcturus PicoPureTM RNA Isolation kit (Arcturus Bioscience), pooled, transcribed to cDNA using random primers and subjected to quantitative real-time polymerase chain reaction (qRT-PCR; SybrGreen Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA)). Primer sequences are listed in Table 1
. The reaction was performed in triplicate with the ABI PRISM 7500HT real-time PCR cycler (Applied Biosystems). Expression of analyzed genes was normalized to the average expression of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT). Expression in injured media and neointima tissue was compared to media of the corresponding noninjured side of the same animals and is shown as mean ± SD.
|
Immunohistochemistry
Sections were deparaffinized (xylene and EtOH) and antigen retrieval was performed by boiling 2 x 7 min at 650 W in a microwave oven using target retrieval solution, high pH (DakoCytomation, Sweden). After washing in PBS-Tween (0.1%) the endogenous peroxidase was blocked by incubation in 3% H2O2 in PBS-Tween (0.1%) for 10 min. For proliferating cell nuclear antigen (PCNA) stainings, sections were then blocked in 20% goat serum 30 min at RT and incubated with primary antibody (Ab), mouse monoclonal anti-PCNA (DakoCytomation) at a dilution of 1:800, over night at 4°C. For BrdU quantification sections of the vessels were incubated with the primary mouse Ab against BrdU (DakoCytomation) at a dilution of 1:50 in PBS containing 1% BSA and 5% horse serum. The BrdU-positive nuclei were counted in the tunica intima and tunica media, and the percentage of positively stained nuclei was determined. The polyclonal rabbit Ab ab16868 recognizing the pY1021-site at the phosphorylated PDGF-ß receptor was purchased from Abcam (Cambridge, UK), the polyclonal rabbit Ab recognizing the PDGF-ß receptor (CTß) was in-house-derived and raised against the glutathione S-transferase (GST)-fusion protein containing the carboxy-terminal part of the human PDGF-ß receptor. Biotinylated goat anti-mouse or anti-rabbit IgG (1:1000, DakoCytomation) were used as secondary antibodies. Furthermore, fixed paraffin-embedded transfected porcine aortic endothelial (PAE) cells overexpressing either the (PAE cells) or the PDGF-ß receptor (ßPAE cells) were used to verify the specificity of the CTß and pY1021 antibodies. PAE and ßPAE cells were serum deprived and stimulated with PDGF-BB (100 ng/ml, 1 h on ice), prior to fixation and paraffin embedding. In the analyses of tissue sections, sections were incubated with preimmune rabbit IgG as a negative control. For quantification of phosphorylated PDGF-ß receptors in vivo, images were digitized, and the staining intensity of ab16868 was quantified on an arbitrary scale between 1 and 10 by two investigators blinded to the treatment protocol. The quantifications of the two investigators of each individual section never varied more than 1 on the arbitrary scale.
Immunoprecipitation and immunoblotting
Vascular smooth muscle cells from the rat aorta isolated enzymatically or by explant technique, DEP-1 knockout mouse embryo fibroblasts (MEFs) (generously provided by Tamás Csikós and Anton Berns, Netherlands Cancer Institute) and wild-type (WT) MEFs were grown to subconfluence, synchronized by serum-deprivation, and left resting or stimulated with either PDGF-BB (10 ng/ml) for 5 min. Pervanadate (100 µM) was given to cells 5 min prior to PDGF stimulation, or for 10 min without PDGF stimulation. Cells were lysed in lysis buffer (20 mM Tris, pH 7.5/1% Nonidet P-40/10% glycerol/1 mM benzamidine/1% Trasylol). The PDGF-ß receptor was collected by 40 µl (1:1 slurry) wheat-germ-agglutinin-sepharose (WGA-sepharose, Amersham, Uppsala, Sweden) or immunoprecipitated with 2 µg CTß at 4°C end-over-end for 1 h. The immunoprecipitates were collected by 40 µl (1:1 slurry) protein A sepharose (Amersham) end-over-end for 1 h. The precipitates were washed 3 times in lysis buffer and resuspended in 2x SDS-sample buffer. Immunoblotting was performed with standard protocols using CTß, PY99 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for detection of phosphotyrosines or PY1021 (Abcam) as primary antibodies. For detection of p42/44 and phospho-p42/44, total cell lysates were resuspended in 2x SDS-sample buffer, followed by standard immunoblotting procedures with antisera recognizing total MAP kinase p42/44, and p42/44 phosphorylated at Thr202/Tyr204 obtained from Cell Signaling Technologies (Beverly, MA, USA). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham) were diluted to 1:25,000 in TBS/0.05% Tween and filters were incubated for 1 h at room temperature. Enhanced chemiluminescence (ECL) (Amersham) was used for visualization.
Protein tyrosine phosphatase activity
For analyses of phosphatase activity in tissue, sections derived from balloon-injured carotid arteries and the corresponding contralateral uninjured arteries from the same animals (500 µm/animal) were pooled in lysis buffer without Na3VO4 generating four different pools of lysates (8 and 14 days balloon-injured arteries, 8 and 14 days uninjured control-side of the same animals). Phosphatase activity was determined by using a 32P-labeled peptide as substrate. Assays were performed in duplicate, and phosphatase activity was determined as the amount of 32P-labeled radioactivity released from the peptide after 7 min of incubation at 30°C and was normalized to protein concentration.
ELISA
Five hundred micrometers/artery were sectioned and pooled according to the four different animal groups (8 and 14 days balloon-injured arteries, 8 and 14 days uninjured contralateral control-arteries side of the same animals). After adjustment for protein concentration, the same amount of proteins was subjected to an ELISA, determining the tyrosine phosphorylation of the PDGF-ß receptor at site pY751 (PathScan® (R) Phospho-platelet-derived growth factor receptor beta (Tyr751) Sandwich ELISA Kit, Cell Signaling, MA, USA). Results of vessels derived from balloon-injured carotid arteries were then normalized to p751-phosphorylation of the corresponding uninjured contralateral control-side arteries. The same procedure as described above was used for determination of the expression of the PDGF-ß receptor. Instead of using the pY751-antibody provided in the kit, a mouse-anti PDGF-ß receptor Ab (clone PR7212, Calbiochem, San Diego, CA, USA), was used.
Chemotaxis and proliferation assays
PDGF-dependent chemotaxis was assayed using a modified Boyden chamber with filters of a pore size of 8.0 µm (Neuroprobe, Gaithersburg, MD, USA) (22)
. Briefly, 10,000 MEF WT and knockout cells were allowed to migrate through collagen I-coated plates for 4 h toward a PDGF-BB (10 ng/ml) gradient. Nonmigrated cells were gently removed from the upper surface, and migrated cells were fixed and stained with Diff-Quick (Dade Behring AG, Düdingen, Switzerland) or with Giemsa (VWR International AB, Stockholm, Sweden). Chemotaxis was quantified by counting the number of migrated cells in one representative high power field in each well. Experiments were carried out with 4–8 wells per condition and performed five times. PDGF-dependent cell cycle progression was measured by a 5-bromodeoxyuridine-incorporation assay. Briefly, 10,000 MEF WT and knockout cells were synchronized for at least 12 h, and PDGF-BB (10 ng/ml) was added to cells for 2 h. BrdU-incorporation was measured according to the manufacturer’s specifications (Roche, Mannheim, Germany) with an incorporation time of 16 h.
siRNA transfection
VSMCs were maintained at 37°C in humidified air with 5% CO2 in Dulbecco’s modified Eagle medium (Sigma) containing 10% FBS. For optimization of siRNA transfection, different transfection procedures (cell concentration, time of transfection), and siRNA concentrations were used to deliver DEP-1 targeting siRNAs into VSMCs. Transfection of 300,000 cells in 6-well plates for 72 h and 100 nM siRNA of four different DEP-1 targeting sequences (Dharmacon, Lafayette, CO) gave the best down-regulation of DEP-1 on mRNA level and therefore was used in subsequent experiments. Cells transfected with chemically unmodified nontargeting control siRNA, and mock-transfected cells served as a control for nonsequence-specific effects of these molecules. Transfection was carried out according the manufactures (Dharmacon) recommendations.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A). Lesions were quantified with regard to morphometrical changes. As shown in Fig. 1B
, the ratio of neointima and media was highest at the late time point. This increase occurred entirely through growth of the neointima (area 0.137±0.021 vs. 0.226±0.110, 8 days vs. 14 days after balloon-injury), whereas the size of the media did not vary over time (area 0.206±0.009 vs. 0.208±0.030, 8 days vs. 14 days after balloon-injury).
|
The lesions obtained from animals 8 and 14 days after induction of neointima formation, and uninjured control vessels were analyzed with regard to VSMC proliferation by immunohistochemical analyses of BrdU incorporation and PCNA expression. Proliferation patterns thus displayed clear time-dependency. Quantifications revealed that BrdU incorporation was 
3- and 2-fold higher at day 8 in the media and neointima, respectively, as compared to day 14 (also see Fig. 2
B). These findings are in agreement with previous analyses of this model.
|
Laser capture microdissection combined with quantitative real-time PCR as a novel approach for layer-specific analyses of gene expression in neointima
Laser capture microdissection was applied as a novel approach to quantitatively describe gene expression in injury-induced lesions in situ. Figure 2A
depicts an example of layer-specific isolation of tissue, through laser-capture microdissection, from areas of interest. RNA was extracted from this isolated material, reverse transcribed to cDNA and used as a template for qRT-PCR analyses.
To validate the method, we analyzed the temporal and spatial expression of the proliferation marker Ki67 in the media and neointima, 8 and 14 days after balloon injury and compared these to results obtained after quantification of BrdU incorporation (Fig. 2B
). The results from the immunohistochemically based BrdU analyses of cell proliferation and of the qRT-PCR-based analyses of Ki67 expression using RNA derived from the microdissected tissue, yielded very similar results. Both methods of analyses indicated higher proliferation in media and intima at day 8 as compared to day 14 and, in general, higher proliferation in the neointima than in the media.
Dynamics of PDGF receptor expression and phosphorylation after balloon injury
The cDNA generated from mRNA obtained from the microdissected vessel layers was used to analyze the dynamics of PDGF- and PDGF-ß receptor expression during lesion formation by qRT-PCR. Both receptors displayed moderately increased expression in the media at 8 days, whereas stronger up-regulation was observed in both media and neointima at the 14-day time point, as compared to control tissue (Fig. 3
A). Because PDGF-ß receptors have been identified as the major mediators of PDGF-dependent neointima formation (23)
, we further focused on the expression and activation of this receptor.
|
The expression pattern of the PDGF-ß receptor protein was analyzed with novel PDGF-ß receptor antibodies. The specificity of these antibodies was confirmed by analyses of fixed and paraffin-embedded cultured cells, which showed strong staining of PDGF-ß receptor expressing porcine aortic endothelial cells (ßPAE cells), whereas PAE cells expressing the PDGF- receptor (PAE cells) were not stained (Fig. 3B
). The immunohistochemical analyses of PDGF-ß receptor in the injured vessels showed highest expression at day 14 in both layers (Fig. 3C
), and a slight up-regulation in the media at the earlier time point, in agreement with the transcript expression analyses (Fig. 3A
).
PDGF-ß receptor phosphorylation was first analyzed by immunohistochemical analyses using antibodies recognizing the pY1021-site of the activated PDGF-ß receptor. The specificity of the Ab was verified with sections of paraffin-embedded nonstimulated and ligand-stimulated PAE and ßPAE cells. Only ßPAE cells that were stimulated with PDGF-BB displayed positive staining patterns (Fig. 3D
). Analyses of the lesions revealed time-and layer-specific differences in PDGF-ß receptor phosphorylation (Figs. 3E and F
). In both layers, receptor phosphorylation was highest at day 8. When media and neointima were compared at this time point, a stronger signal was observed in the neointima.
In a second approach of determining PDGF receptor phosphorylation, an ELISA method was used to analyze PDGF receptor phosphorylation in vessel extracts. As shown in Fig. 3G
, this method confirmed that receptor phosphorylation was reduced at day 14, as compared to day 8. This aspect was further emphasized when results were normalized to receptor expression levels.
Together, these analyses thus indicate a strong correlation between the dynamics of the PDGF-ß receptor phosphorylation and the VSMC proliferation (Figs. 1B
, 2C
, and 3E-G
).
Expression pattern of PDGF isoforms and PDGF-ß receptor-targeting PTPs in the vessel wall
Since the correlation of PDGF-ß receptor phosphorylation with the proliferation pattern in the injured tissues could not simply be explained by the expression level of the receptors, we next explored other components of PDGF signaling. Therefore, the expression of all four PDGF isoforms and the endogenous antagonists of PDGF-ß receptor signaling, protein tyrosine phosphatases (PTPs) were analyzed.
Only two of the four PDGF isoforms, PDGF-A and PDGF-C, showed a moderate up-regulation in the media layer at the 8-day time point, whereas neither ligand was up-regulated at the early time point in the neointima, as compared to control media (Fig. 4
A). However, at day 14, all four isoforms were up-regulated. PDGF-B showed a more than five-fold up-regulation in the neointima, whereas all isoforms were characterized by a two- to threefold up-regulation in both media and neointima. Taken together, these results do not provide evidence for simple and direct relationships between the levels of ligand expression and receptor phosphorylation.
|
To explore whether PDGF-ß receptor-targeting PTPs contribute to the pattern of PDGF-ß receptor phosphorylation (Fig. 3E and 3F
) and VSMC proliferation (Figs. 1C
and 2B)
, we analyzed the expression of five PTPs that have been described to interact with the PDGF-ß receptor (10
, 24)
: protein tyrosine phosphatase-1B (PTP-1B), T cell protein tyrosine phosphatase (TC-PTP), density-enhanced protein tyrosine phosphatase (DEP-1), receptor-like protein tyrosine phosphatase alpha (PTP), and src homology domain 2-containing protein tyrosine phosphatase 2 (SHP-2) (Fig. 4B
). The neointima at day 8, characterized by high receptor phosphorylation and high VSMC proliferation, displayed 10-fold lower expression as compared to control media (Fig. 4B
, right). In contrast, the same vessel layer at day 14, displaying low receptor PDGF-ß phosphorylation and low proliferation despite abundant ligand and receptor expression, was characterized by a distinct up-regulation of TC-PTP, PTP-1B, and PTP, which occurred concomitant with a normalization of DEP-1 levels (Fig. 4B
, right). A similar pattern was also observed in the low-proliferative media of day 14 (Fig. 4B
, left). The media at day 8, characterized by a moderate increase in proliferation, displayed an intermediate pattern with an approximate 1.5–2-fold up-regulation of TC-PTP, PTP-1B, and PTP, and a twofold down-regulation of DEP-1. Interestingly, SHP-2, which is the only one of the analyzed PTPs that has not been demonstrated to antagonize PDGF ß-receptor signaling, was characterized by no, or a very moderate, regulation in its expression (Fig. 4B
, left and right).
Together, these findings demonstrated that vessel expression of PTP mRNAs was highest at the late time point. To investigate whether this was paralleled by an increase in PTP activity, total vessel extracts were prepared and characterized in an in vitro PTP assay (Fig. 4C
). In agreement with the mRNA analyses, the PTP activity was significantly higher in lesions, as compared to control vessels, at the late time point. At day 8 after injury, a moderate reduction was seen, which is compatible with the mRNA expression which indicated smaller increases in TC-PTP and PTP-1B, concomitant with larger reductions of DEP expression.
In general, the expression of PDGF-ß receptor-antagonizing PTPs thus showed an intriguingly inverse expression pattern to that of the PDGF-ß receptor phosphorylation and VSMC proliferation. Because DEP-1 expression displayed a striking inverse relationship to both PDGF-ß receptor activation and VSMC proliferation in vivo, we further investigated the possible impact of DEP-1 on PDGF signaling.
PDGF-ß receptor signaling in DEP-1-deprived fibroblasts and VSMCs
To preliminarily analyze the contribution of PTPs in controlling phosphorylation of PDGF-ß receptors in VSMCs, receptor phosphorylation was analyzed after treatment of VSMCs with the general PTP inhibitor pervanadate (Fig. 5
A). High phosphorylation of the PDGF-ß receptor, which was not further increased by additional PDGF-BB-treatment, was seen after pervanadate treatment of VSMCs (Fig. 5A
), clearly demonstrating a major role of PTPs in regulation of the levels of PDGF-ß receptor phosphorylation in VSMCs.
|
PDGF-ß receptor phosphorylation was also compared in mouse embryo fibroblasts (MEFs) derived from WT mice and DEP-1–/– mice (Fig. 5B
, left). Increased PDGF-ß receptor phosphorylation, as determined by immunoblotting with phospho-tyrosine-antibodies or antibodies recognizing the DEP-1 dephosphorylation site pY1021 of the PDGF-ß receptor (25)
, was observed in DEP-1–/– MEFs (Fig. 5B
left). Moreover, this increase in receptor phosphorylation was accompanied by a moderate increase of phosphorylation of the PDGF-ß receptor downstream-signaling molecule p42/44 in knockout cells compared to WT MEFs (Fig. 5B
, right). Quantification of results from four experiments indicated an approximately twofold increase in p42/44 phosphorylation in PDGF-stimulated DEP-1 –/– cells, as compared to stimulated control cells (data not shown). Furthermore, DEP-1 –/– cells also displayed an increased PDGF-induced Akt phosphorylation as compared to control cells (data not shown).
Proliferation, determined by BrdU-incorporation, of DEP-1–/– MEFs and WT cells was complicated to evaluate because of low response in WT MEFs but showed a trend toward increased PDGF-BB-induced incorporation in knockout cells (Fig. 5C
, left). However, chemotaxis, another cellular response relevant for PDGF-dependent neointima formation, was significantly enhanced in PDGF-BB-treated DEP-1–/– cells when compared to WT MEFs (Fig. 5C
, right). The chemotactic response of DEP-1–/– cells was strongly inhibited by pharmacological blockage of p42/p44 signaling through addition of PD98059 (data not shown).
To experimentally substantiate the notion of DEP-1 as a PDGF-ß receptor-antagonizing PTP also in VSMCs, DEP-1 was down-regulated in cultured VSMCs by siRNA. DEP-1 siRNA-transfections specifically reduced DEP-1 expression to 40% of that seen in control cells (Fig. 6
A). Down-regulation of DEP-1 in VSMCs led to a moderate increase in ligand-dependent PDGF-ß receptor phosphorylation (Fig. 6B
, top). A clear enhancement in phosphorylation of p42/44 was also observed in siRNA-mediated DEP-1 down-regulated VSMCs after PDGF-BB stimulation (Fig. 6B
, bottom). Furthermore, overexpression of DEP-1 in VSMCs, following transfection with DEP-1 expression vectors, reduced ligand-induced PDGF receptor phosphorylation (data not shown).
|
Thus, the results observed in DEP-1–/– MEFs and in VSMCs, in which DEP-1 had been down-regulated by siRNA, provide further support for a causal relationship between the DEP-1 down-regulation and the increased PDGF-ß receptor phosphorylation observed at day 8 in the media and neointima of injured vessels.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, thus strongly suggest a previously unrecognized role of dynamically expressed PTPs in control of PDGF-dependent neointima growth. In addition, the study identified DEP-1 as a novel physiological regulator of PDGF receptor signaling based on analyses of DEP-1–/– cells and VSMCs treated with DEP-1 siRNA. Finally, the detailed quantifications of proliferation, expression and phosphorylation of PDGF receptors, as well as PTP expression, clearly show that the largest changes over time are observed in the neointima and thus confirm that in this animal model the neointima is the most dynamic vessel layer.
Previous characterization of PDGF receptor signaling in knockout fibroblast has identified also TC-PTP and PTP-1B as PDGF receptor antagonizing PTPs (6
, 8)
. In contrast, analyses of fibroblasts lacking PTP-
failed to detect changes in PDGF signaling (6)
. Together, these studies thus establish that multiple, but not all, PTPs are involved in the control of PDGF receptor phosphorylation. Given the fact that many of these PTPs also act on other receptor tyrosine kinases, it is predicted that the observed changes in PTP expression will also impact on the signaling of other receptors expressed on VSMCs.
The dynamics of PTP expression during pathological RTK-dependent tissue remodeling have not previously been extensively analyzed. However, studies on developmental processes have clearly demonstrated the importance of transcriptionally regulated PTPs as modulators of RTK signaling. For example, Berset and coworkers identified the Caenorhabditis elegans homolog of DEP-1 as a negative regulator of the epidermal growth factor (EGF) receptor during vulva development, in a study which demonstrated that highly specific spatial and temporal regulation of DEP-1 expression was required for binary cell fate decisions and subsequent proper vulva development (11)
. In addition, CLR-1, another receptor-like PTP has been identified as a functional antagonist of FGF receptor-regulated developmental processes in C. elegans (26)
. To analyze if similar dynamics are involved in RTK-dependent pathological processes and in mammalian development, obviously, merits further studies.
The principal mechanisms and molecular details of the transcriptional regulation of PTPs, in general, and DEP-1, in particular, are largely unknown. Recent findings include identification of the Y box-binding protein YB-1 as an important regulator of PTP-1B (12)
expression, and evidence that DEP-1 expression in T-cells is regulated by T cell receptor activation (27)
. It is also noteworthy that stimulation of cultured VSMCs with PDGF, or FGF, increased the expression of both PTP-PEST and PTP-1B (19)
. To what extent PTP induction by RTK ligands represents a general mechanism for feedback inhibition of RTK signaling is thus an obvious and relevant topic for future studies.
This study used laser capture microdissection (LCM) as a novel method for analysis of in situ gene regulation in vessel biology. LCM yielded reliable material from its in situ environment, as validated by the concordance of immunohistochemical analyses and qRT-PCR analyses of the expression of Ki67 and PDGF-ß receptor (Figs. 2
and 3)
. Despite the limitation of monitoring mRNA expression, rather than protein levels, this method still holds promise for many additional applications in the analyses also of other aspects of vessel biology. As compared to immunohistochemistry, which is restricted by the availability and quality of antibodies, LCM has the clear advantages of allowing simultaneous, and, in particular, quantitative, analyses of multiple genes in the same tissue preparations.
From a therapeutic perspective, our study strengthens the notion of PTP activation as a valid strategy for pharmaceutical interference. Experimental support for this general concept was recently provided by the demonstration of a reduction in PDGF-BB-induced proliferation and migration following overexpression of PTP-1B in cultured VSMCs (20)
. Continued detailed analyses of the involvement of antagonistic PTPs in the regulation of RTK-driven pathologies is thus predicted to pave the way for PTP-activation as a yet unexploited therapeutic strategy for restenosis and other diseases involving RTK-driven tissue remodeling.
|
ACKNOWLEDGMENTS |
|---|
Received for publication August 16, 2006. Accepted for publication September 13, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Karagyozov, R. Godfrey, S.-A. Bohmer, A. Petermann, S. Holters, A. Ostman, and F.-D. Bohmer The structure of the 5'-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation Nucleic Acids Res., August 1, 2008; 36(13): 4443 - 4453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Pilecka, C. Patrignani, R. Pescini, M.-L. Curchod, D. Perrin, Y. Xue, J. Yasenchak, A. Clark, M. C. Magnone, P. Zaratin, et al. Protein-tyrosine Phosphatase H1 Controls Growth Hormone Receptor Signaling and Systemic Growth J. Biol. Chem., November 30, 2007; 282(48): 35405 - 35415. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
