Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G1/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D1 expression, as well as p27Kip upregulation. Cell division kinase 2 (cdc2) phosphorylation at G2/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.
- tyrosine kinase
- protein synthesis
- cell cycle
- protein kinase B
- extracellular signal-regulated kinases 1 and 2
in the normal vessel wall, vascular smooth muscle cells (VSMC) are highly differentiated and exhibit a very low proliferation rate (26). In response to changes in the neurohormonal milieu or injury to the vessel wall, VSMC dedifferentiate into a hyperplastic synthetic phenotype, characterized by increased proliferation, hypertrophy, and migration (39). This aberrant smooth muscle cell growth contributes to the progression of cardiovascular disease, particularly atherosclerosis and balloon angioplasty-induced restenosis (14, 30, 31). The regulation of VSMC growth is influenced by a variety of growth factors, mechanical forces (11), oxidative stress (40), and the renin-angiotensin system (RAS) (8, 10, 45). Growth factors are potent mitogens produced locally within the blood vessel wall and play a crucial role in the development of atherosclerosis and neointima formation following vascular injury (1). Among these growth factors are platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast-derived growth factor (FGF), and the insulin-like growth factor I (IGF-1) (37, 55).
Regulation of VSMC growth requires interplay between a complex series of events, including immediate early gene expression, protein and DNA synthesis, and assembly of newly synthesized cytoskeletal components (4). Synchronized cell cycle control is essential for VSMC proliferation while coordinated regulation of protein synthesis is crucial for hypertrophic growth. Upon stimulation, quiescent VSMC enter the G1 phase, allowing for the synthesis of mRNA and proteins necessary for DNA synthesis in ensuing S phase. The regulation of G1 progression and G1/S transition is governed by a delicate interplay of cyclins, cyclin-dependent kinases (cdks), and their inhibitors (cdkI) such as p21waf1 and p27kip (6, 9). Cyclins D and E, which are rapidly synthesized during G1, bind to cdk4 and cdk2, respectively, to mediate G1/S transition and DNA synthesis (41, 48). The retinoblastoma protein (Rb) is a major target of cdk4/6. Upon phosphorylation, Rb dissociates from the adenoviral E2-motif-binding transcription factor (E2F) transcription factor, enabling E2F to initiate gene transcription (19, 24, 50). p21waf1 and p27kip are important negative regulators of cyclin/cdk interactions required for Rb phosphorylation. p21waf1 is upregulated by p53 and inhibits cdk4/6 activity (21), whereas p27kip inhibits cdk2 activity, thus preventing entry into the S phase (29, 41, 56). Both angiotensin II (ANG II) and PDGF have been shown to regulate several key proteins that are involved in G1/S transition, including expression of cyclins D and E and activation of cdk4. PDGF induces G1/S transition through cdk2 activation, which correlates with a dramatic decrease in p27kip expression.
Transition through the G2/M checkpoint requires complex formation between cdc2 (cell division 2 kinase) and cyclin B to form the “mitosis promoting factor.” cdc2 activation requires phosphorylation on Thr161 by cdk-activating kinase (CAK) and dephosphorylation of Tyr15 by cdc25 phosphatase (51).
The PDGF-induced progression of the cell cycle in VSMC is regulated through multiple signaling cascades, including mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway (7, 20, 21, 52). Nonreceptor proline-rich tyrosine kinase 2 (PYK2) plays an important role in AT1-receptor-mediated signaling (5, 42–46). However, the participation of PYK2 in growth factor receptor signaling, particularly tyrosine kinase receptor signaling (e.g., PDGF), remains to be elucidated. Recent evidence from our own studies and others has shown that PYK2-induced AKT activation is an important mediator of ANG II-induced protein translation (12, 42, 44, 45, 53). PYK2 is activated in PDGF-induced VSMC migration, together with extracellular-regulated kinases 1 and 2 (ERK1/2) MAPK. However, the role of PYK2 in PDGF-induced cell cycle progression is not yet fully understood. In the present study we address these issues and determine the specific effects of PDGF-stimulated PYK2, AKT, and ERK1/2 activation on PDGF-dependent cell proliferation. Therefore, the present study examines specific effects of PDGF-stimulated PYK2 activation on signaling targets (MAPK and AKT) and identifies a role for PYK2 in PDGF-dependent cell proliferation.
MATERIALS AND METHODS
PYK2 antisense oligodeoxynucleotides were custom designed by Biognostik (Göttingen, Germany). Custom-designed PYK2 small interfering RNA (siRNA) (SMARTpool) was from Dharmacon. Anti-total PYK2 and anti-phospho PYK2 (pTyr402) were from Transduction Laboratories. Anti-total c-Fos antibodies were from Santa Cruz Biotechnology. Anti-phospho ERK1/2 (pThr-202/Tyr-204), anti-phospho AKT (pThr−308), anti-phospho AKT (pSer−473), anti-phospho p38MAPK (pThr180/Tyr182), anti-total AKT, anti-total (ERK1/2), anti-total p38MAPK, anti-phospho Rb (pSer807/811, pSer795, pSer780), anti-phospho cdc2 (pThr161), anti-total cyclin D1, anti-total p27kip, and anti-total β-actin antibodies were from Cell Signaling. [3H]phenylalanine and [3H]thymidine were purchased from Amersham Pharmacia Biotech. PI3K inhibitor LY-294002 and MEK-1 inhibitor U-0126 were purchased from Calbiochem. LipofectAMINE (LFN) Plus was purchased from Life Technologies. DharmaFECT1 was from Dharmacon. PDGF-BB was purchased from R&D Systems.
Cell culture and treatments.
VSMC were prepared and cultured as previously described (27). In all cases, cells were growth arrested in 0.2% CS-DMEM overnight, and medium was changed to that respective for each experiment at least 1 h before treatment. Pretreatment with PI3K inhibitor LY-294002 or MEK inhibitor U-0126 was performed 45 min before addition of PDGF (10 ng/ml) for specified times.
PYK2 antisense oligonucleotide and PYK2 siRNA incorporation.
VSMC were grown in 10% CS-DMEM to ∼60% confluence. Cells were washed three times in Opti-MEM medium (Life Technologies) 1 h before antisense or siRNA treatment. VSMC were treated with PYK2 antisense oligodeoxynucleotides (0.75 μM) for 8 h (45). LipofectAMINE Plus was used as a transfection reagent. After 8 h, the medium was replaced with 0.2% CS-DMEM and left overnight. The next day 0.2% CS-DMEM was replaced with serum-free DMEM for at least 1 h before treatment with PDGF. For siRNA treatment, VSMC were placed in 0.2% CS-DMEM and treated with PYK2 siRNA or nontargeting scrambled control siRNA for 24 h (100 nM) and then transferred to serum-free DMEM for 2 h before treatment with PDGF. DharmaFECT 1 (for siRNA) was used as a transfection reagent.
Protein synthesis measurements.
VSMC were treated with PYK2 antisense oligodeoxynucleotides as described above. The next day 0.2% CS-DMEM was replaced with serum-free DMEM for at least 1 h before stimulation with 10 ng/ml PDGF for 24 h. During the last 6 h of PDGF incubation, 1 μCi/ml [3H]phenylalanine (30 Ci/mmol) was added to each dish. Total protein measurements were assessed at the end of the 24-h incubation period. VSMC were rinsed with 1 ml of ice-cold phosphate-buffered saline, and protein was precipitated by 10% trichloroacetic acid for 30 min on ice. The trichloroacetic acid-precipitable material was solubilized with 0.2 M NaOH for 20 min at 60°C. A portion of the sample was used to determine total protein using a bicinchoninic acid (Pierce) protein assay, and [3H]phenylalanine was determined by liquid scintillation counting. Triplicate dishes were used for each measurement.
DNA synthesis measurements.
VSMC were treated with PYK2 antisense oligodeoxynucleotides as described above. The next day 0.2% CS-DMEM was replaced with serum-free DMEM for at least 1 h before stimulation with 10 ng/ml PDGF for 24 h. During the last 6 h of PDGF incubation, 1 μCi/ml [3H]thymidine was added to each dish. After incubation, VSMC were rinsed with 1 ml of ice-cold phosphate-buffered saline and then lightly trypsinized to determine cell numbers in each dish. Cell suspensions were washed twice with 5% trichloroacetic acid and then solubilized by adding 0.5 ml 0.25 N NaOH. [3H]thymidine was determined by liquid scintillation counting, and data are expressed as cpm/106 cells. Triplicate dishes were used in each experiment.
Western blot analyses.
Cell lysates were prepared as described previously (46), and protein from lysates was calculated by bicinchoninic acid protein assay (Pierce). Equal amounts of protein (35 or 75 μg) were resolved by 10 or 12% SDS-PAGE and transferred to nitrocellulose. Immunoblot analyses were performed using antibodies listed above. Bands were visualized by enhanced chemiluminescence (ECL; Amersham) and quantified using NIH Image software.
Data are presented as means ± SE for at least n = 3 experiments. One-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's test was used for comparisons among multiple groups. Differences among means were considered significant at P < 0.05. Data were analyzed using InStat statistical software (GraphPad).
Effect of PYK2 antisense oligodeoxynucleotides on PDGF-induced protein and DNA synthesis.
We previously reported that the PYK2 antisense (AS) oligodeoxynucleotides (PYK2-AS) treatment protocol resulted in ∼80% knockdown of endogenous PYK2 (total protein detected by WB was ∼0.20 ± 0.06-fold of control) with no change in expression of the closely related focal adhesion kinase (FAK) (42). We also previously demonstrated that scrambled oligodeoxynucleotides (S-AS) have no effect on PYK2 or FAK levels and have no effect on VSMC protein or DNA synthesis. Therefore, we first examined the role of PYK2 on PDGF-induced protein synthesis by downregulating PYK2 using AS technology (PYK2-AS). Rat aortic smooth muscle cells (RASMC) were treated with transfection reagent (LFN; 10 μg/ml), S-AS (0.75 μM), or PYK2-AS (0.75 μM), followed by exposure to PDGF (10 ng/ml) in the presence of [3H]phenylalanine during the last 6 h of the 24-h treatment. PDGF induced an approximately twofold increase in protein synthesis that was completely abrogated by PYK2-AS (Fig. 1A).
To determine the effect of PYK2 downregulation on PDGF-induced DNA synthesis, RASMC were treated with transfection reagent (LFN; 10 μg/ml) or PYK2-AS (0.75 μM) followed by PDGF (10 ng/ml) in the presence of [3H]thymidine during the last 6 h of a 24-h treatment. PDGF stimulated a significant increase in DNA synthesis as measured by [3H]thymidine incorporation, which was significantly inhibited by PYK2 antisense oligodeoxynucleotides (Fig. 1B). We previously demonstrated that scrambled oligodeoxynucleotide treatment had no effect on PYK2 expression (42).
Effect of PDGF on cell cycle regulators at the G1/S and G2/M transition.
To examine the effect of PDGF (10 ng/ml) on G1/S and G2/M transition proteins, we probed by Western blot analysis for Rb phosphorylation, p27kip, and cyclin D1 as markers of G1/S and cdc2 phosphorylation as a marker of G2/M transition (Fig. 2). We detected low levels of basal Rb phosphorylation in unstimulated cell lysates [multiple cross-reacting bands that represent different degrees of protein phosphorylation (18)]. A 12-h treatment with PDGF resulted in an approximately threefold increase in overall Rb phosphorylation variants. This increase in phosphorylation remained significant at 18 h and decreased toward basal levels by 24 h (Fig. 2A). The expression of p27kip is inversely correlated with cell cycle progression. Treatment of RASMC with PDGF resulted in decreased p27kip protein levels at 18 h and almost undetectable protein levels at 24 h (Fig. 2B). Cyclin D1 protein increased significantly by 18 h and remained stable at 24 h (Fig. 2C). In Fig. 2D, a significant increase in cdc2 phosphorylation was detected as early as 12 h and further increased at 18 and 24 h.
Effect of PYK2 downregulation on PDGF-dependent regulation of the G1/S and G2/M transitions.
We next investigated the effect of PYK2 downregulation by siRNA on Rb and cdc2 phosphorylation and p27kip and cyclin D1 expression. Treatment with PYK2 siRNA results in a ∼80% knockdown of endogenous PYK2 levels (Fig. 2E) but does not affect the levels of FAK (data not shown). Compared with PDGF treatment, PYK2 siRNA significantly prevented Rb phosphorylation at 12 and 18 h, cyclin D1 expression at 18 and 24 h, as well as cdc2 phosphorylation at 12, 18, and 24 h. Also, PYK2 siRNA prevented the PDGF-induced decrease in p27kip expression at 18 and 24 h. Thus PYK2 downregulation prevented the effect of PDGF on the expression or activation of these cell cycle regulators (Fig. 2). Scrambled control siRNA had no effect on the PDGF-mediated effects on cell cycle proteins at any of the time points examined (data not shown).
PDGF regulates PYK2, AKT, and MAPK phosphorylation.
To determine the effect of PDGF treatment on the activation of various signaling cascades, we examined PYK2, AKT, ERK1/2, and p38 MAPK phosphorylation when RASMC were exposed to PDGF (10 ng/ml) for the times shown. Western blot analysis with anti-phospho-antibodies was used to determine phosphorylation as a surrogate marker for activation. PYK2 phosphorylation was biphasic: a rapid activation that returned near baseline levels by 60 min (Fig. 3A) but then increased at 2 and 4 h (data not shown). PDGF caused a robust phosphorylation of all other kinases examined at 5 min; however, the kinetics of activation differed: ERK1/2 and AKT phosphorylation were sustained at 60 min, whereas p38 MAPK showed a much more transient phosphorylation that returned to baseline at 30 min (Fig. 3, B–D).
Effect of PYK2 antisense oligodeoxynucleotides on PDGF-induced AKT, ERK1/2, and p38 MAPK phosphorylation.
PYK2-AS (0.75 μM) significantly abrogated PDGF-dependent AKT phosphorylation at both Ser473 and Thr308. ERK1/2 phosphorylation was also prevented by PYK2-AS in response to PDGF (10 ng/ml) treatment for 10 min. Conversely PYK2 downregulation had no effect on p38 MAPK phosphorylation (Fig. 4). No changes were observed in the levels of total AKT, ERK1/2, and p38 MAP kinases (not shown).
Effect of PI3K inhibition on PDGF-dependent regulation of the G1/S and G2/M transition.
To investigate the requirement for PI3K/AKT signaling for G1/S or G2/M transition in RASMC, we tested the effect of PI3K inhibition on cell cycle proteins at 18 h. In preliminary studies we found that pretreatment with LY-294002 completely inhibits PDGF-induced AKT activation (data not shown). We also found 18 h to be an optimal timepoint for examining the effect of PI3K signaling on cell cycle proteins at G1/S. VSMC were pretreated with the PI3K inhibitor LY-294002 (5 μM) for 45 min, followed by exposure to PDGF for 18 h. We observed an increase in Rb phosphorylation and cyclin D levels at 18 h in response to PDGF, which was abolished with PI3K inhibition (Fig. 5, A and B). In Fig. 5C, we examined the effect of PI3K inhibition on p27kip protein. PDGF induced a significant decrease in p27kip at 18 h, and this effect was prevented by LY-294002 treatment (Fig. 5C). Moreover, we found that cdc2 phosphorylation (Fig. 5D), an event required for initiation of G2 phase that is induced robustly by PDGF, was blocked by LY-294002.
Effect of MAPK inhibition on PDGF-dependent regulation of the G1/S and G2/M transition.
Since PDGF also causes sustained ERK1/2 activation, we next determined whether the ERK1/2 signaling pathway is involved in PDGF-induced cell cycle progression. Pretreatment with the MEK inhibitor U-0126 (10 μM) completely blocked PDGF-induced ERK1/2 phosphorylation (Fig. 6A), attenuated the PDGF-dependent decrease in p27kip expression (Fig. 6B), and abrogated Rb phosphorylation at Ser807/811 (Fig. 6C). ERK1/2 also appears to regulate PDGF-induced G2/M transition since U-0126 blocked cdc2 phosphorylation at Thr161 (Fig. 6D). Conversely, the p38 MAPK inhibitor SB-203580 had no effect on the PDGF-dependent stimulation of any of the cell cycle proteins examined (data not shown).
PDGF is a potent proliferative agent in VSMC that potentiates changes in cell cycle proteins through the activation of key signaling pathways that regulate cell survival, proliferation, migration, and apoptosis. The results from this study demonstrate a role for PYK2 in PDGF-dependent vascular smooth muscle growth. PYK2 downregulation attenuated PDGF-induced DNA and protein synthesis (Fig. 1) and inhibited downstream signaling pathways involved in the G1/S and G2/M cell cycle progression (Figs. 2 and 4). These results are consistent with data indicating that PDGF-induced VSMC proliferation is associated with increased transit through the G1/S phase of the cell cycle (17).
The molecular mechanisms that link the PDGF receptor to PYK2 are unclear. The normal signaling paradigm for the PDGF receptor involves receptor autophosphorylation and subsequent binding/docking sites for SH2-domain-containing adaptor proteins such as Shc and Grb2 and kinases such as Src, phospholipase C, and PI3K (2, 3). Unlike interaction among PYK2, FGF, and EGF receptor tyrosine kinases (34, 49), we failed to detect an interaction between PYK2 and the PDGF receptor (data not shown). It is possible that Src or Ca2+-dependent protein kinase C isoforms may link PDGF receptor activation to PYK2 as we and others have previously shown for the angiotensin AT1 receptor (5, 36, 44, 46).
Our results indicate that sustained PYK2 phosphorylation by PDGF links the PDGF receptor to downstream signaling cascades including ERK1/2 and PI3K-AKT (Fig. 4), as well as the regulators of the G1/S and G2/M transition and thus cell cycle progression (Fig. 2). This is particularly interesting, especially given the fact that PI3K has direct binding sites on the PDGF receptor, yet there is a requirement for PYK2 for AKT phosphorylation in response to PDGF. We cannot speculate as to the reason for this effect, yet it may warrant further investigation.
Importantly, PDGF caused a robust increase in Rb and cdc2 phosphorylation and expression of cyclin D1, but decreased p27kip1 expression and downregulation of PYK2 expression reversed these effects of PDGF (Fig. 2) and blocked PDGF-induced phosphorylation of AKT and ERK1/2 (Fig. 4). Modulation of these pathways was associated with PDGF-dependent phosphorylation of PYK2 at its autophosphorylation site (Tyr402) (54), suggesting that PYK2 kinase activity may be required for activation. Recently, Lim et al. (23) reported that PYK2 promotes proliferation of endothelial cells and embryonic fibroblasts through downregulation of the tumor suppressor protein p53 in a kinase-independent manner via its four point one ezrin, radix and moesin (FERM) scaffolding domain. Since PDGF downregulates p53 in VSMC (13), we cannot rule out the possibility that a PDGF-PYK2-p53 pathway also contributes the VSMC cell cycle progression and proliferation. Further studies will be required to tease out this interesting interaction and assess the specific role of this tumor suppressor in PDGF-dependent proliferation of RASMC.
Inhibition of PI3K-AKT (LY-294002) and MEK-1-ERK1/2 (U-0126) signaling pathways abrogated the effects of PDGF by preventing Rb and cdc2 phosphorylation, inhibiting the increase in cyclin D1, and preventing the decrease in p27kip (Figs. 5 and 6). These results suggest that ERK1/2 and AKT signaling pathways are involved in the regulation of both G1/S and G2/M transitions. However, we cannot rule out the involvement of other signaling molecules downstream of PI3K. These findings are consistent with our hypothesis that PYK2 mediates PDGF-induced signaling, cell cycle changes, and proliferation and further establish the specific role of these downstream targets of PYK2 in mediating the effects of PDGF for RASMC proliferation.
The PI3K/AKT axis regulates G1/S transition at multiple molecular inputs, including stabilization of cyclin D1 and c-myc through AKT-mediated glycogen synthase kinase-3 β inhibition, increased p27Kip stability, and inhibition of forkhead family transcription factors (22). The role of AKT in the regulation of G2/M transition is less clear; however, it has been shown that AKT directly phosphorylates the DNA damage checkpoint kinase Chk1 on Ser280, resulting in its translocation to the cytosol (28). Since Chk1 can induce cdc25C phosphorylation and sequestration into the cytosol by protein 14-3-3; inhibition of Chk1 allows cdc25C phosphatase to remain in the nucleus and dephosphorylate cdc2 at an inhibitory site by Wee1-like kinases. Chk1 inhibition by AKT family of kinases would indirectly lead to cdc2 activation and induction of the mitosis promoting factor (cdc2/cyclin B complex) required for G2/M transition (22).
Our data are consistent with the role of sustained ERK1/2 activation as a “master regulator” of the G1/S phase transition (see Ref. 33 for a recent review). The mechanism by which ERK1/2 suppresses p27Kip is unclear, but Sakakibara et al. (47) recently reported that posttranscriptional RNA turnover may be involved. The downstream effectors that link ERK1/2 to cell cycle regulation have not been fully characterized. Activation of ERK1/2 can lead to its nuclear translocation and subsequent activation of transcription factors such as Elk-1, to increase gene transcription and protein synthesis (25). More recently, ERK1/2 has been implicated in PDGF-induced upregulation of the neuron-derived orphan receptor-1 in VSMC that is necessary for cyclin D1 expression and cell cycle progression (38).
In contrast to regulating PDGF-dependent ERK1/2 and AKT activation, PYK2 does not appear to be upstream of p38 MAPK since PYK2 antisense had no effect on PDGF-induced p38 phosphorylation (Fig. 4). PDGF-induced G1/S transition was also unaffected by p38 inhibition (data not shown). These findings are consistent with the anti-proliferative effects of p38 MAPK in vascular smooth muscle (16, 35) and mesangial cells (15). Also, in contrast to PDGF-induced responses, PYK2 is an upstream regulator of p38 MAPK activation in response to ANG II, which, alone, cannot induce VSMC proliferation (42).
The present study is the first to examine the role of PYK2 on PDGF-dependent VSMC signaling and proliferation. Our results suggest that VSMC have distinct responses to PDGF that are regulated at the level of signal transduction. We find these results to be of particular relevance since ANG II and PDGF promote different growth effects in VSMC yet activate the same molecules to mediate differential growth responses. From previous studies and our own experiments, we have observed sustained p38 MAPK and transient ERK1/2 and AKT phosphorylation in response to ANG II compared with the opposite effect of PDGF on the duration of activation of the same signaling pathways (sustained ERK1/2 and AKT but transient p38 MAPK activation) (32, 45). Moreover, whereas it has been demonstrated that ANG II-dependent p38 MAPK activation is mediated by PYK2, this study indicates that PYK2 does not mediate PDGF-dependent p38 MAPK activation in response to PDGF. These data support a differential signaling pathway activation mediated by PYK2 in response to these vasoactive agents, which emphasizes the importance of the exquisite level of regulation of MAPK and PI3K signaling pathways in normal cells. These findings raise the possibility that the use of specific inhibitors to block distinct cell signaling pathways can be considered as potential therapeutics to prevent pathological PDGF-induced smooth muscle cell growth. Future studies are required to link upstream mediators and downstream molecular targets of PYK2-dependent signaling pathways regulated by PDGF receptor activation.
This study was supported by grants National Institutes of Health Grants2RO1-HL-5604 and NIH/NCRR 1P20RR18766 (to P. A. Lucchesi) and NIH ES10167 (to V. Darley-Usmar). J. Perez was supported by NIH Training Grant NIH T32 HL-07918.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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