This Article Abstract Full Text (PDF) All Versions of this Article: 22/11/1790    most recent 01.ATV.0000034475.40227.40v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tai, L.-K. Articles by Berk, B. C. Search for Related Content PubMed PubMed Citation Articles by Tai, L.-K. Articles by Berk, B. C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLCYSTEINE CALCIUM COMPOUNDS CALCIUM, ELEMENTAL CYTOCHALASIN D HYDROGEN PEROXIDE Related Collections Cell signalling/signal transduction Endothelium/vascular type/nitric oxide

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1790.)
© 2002 American Heart Association, Inc.

Vascular Biology

Fluid Shear Stress Activates Proline-Rich Tyrosine Kinase via Reactive Oxygen Species–Dependent Pathway

Lung-Kuo Tai^*; Masanori Okuda^*; Jun-ichi Abe; Chen Yan; Bradford C. Berk

From the Center for Cardiovascular Research (L.-K.T., J.A., C.Y., B.C.B.), University of Rochester, Rochester, NY, and the Division of Cardiovascular and Respiratory Medicine (M.O.), Kobe University Graduate School of Medicine, Kobe, Japan.

Correspondence to Bradford C. Berk, MD, PhD, University of Rochester, Department of Medicine, Box MED, Rochester, NY 14642. E-mail bradford_berk{at}urmc.rochester.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— Fluid shear stress (flow) modulates endothelial cell (EC) function via specific signal transduction events. Previously, we showed that flow-mediated tyrosine phosphorylation of p130 Crk-associated substrate (Cas) required calcium-dependent c-Src activation. Because flow increases reactive oxygen species (ROS) production in ECs and because H₂O₂ increases tyrosine phosphorylation of proline-rich tyrosine kinase (PYK2), we hypothesized that flow may activate PYK2 via ROS.

Methods and Results— Exposure of bovine aortic ECs to flow stimulated PYK2 phosphorylation rapidly, with a peak at 2 minutes. The activation of PYK2 and phosphorylation of Cas induced by flow were inhibited by pretreatment with the antioxidant N-acetylcysteine. Flow-induced PYK2 phosphorylation was inhibited by BAPTA-AM, an intracellular calcium chelator. Bovine aortic ECs transfected with kinase-inactive PYK2 showed attenuated flow-stimulated Cas tyrosine phosphorylation. Although flow-induced Cas phosphorylation was inhibited by kinase-inactive Src, PYK2 activation induced by flow was not inhibited by overexpression of kinase-inactive Src.

Conclusions— These results show a redox-sensitive pathway for flow-mediated activation of nonreceptor tyrosine kinase activity that requires ROS and intracellular calcium, but not Src kinase.

Key Words: proline-rich tyrosine kinase • calcium • reactive oxygen species • shear stress

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Vascular endothelial cells (ECs), which form the inner lining of the blood vessel wall, play an important role in the regulation of vascular homeostasis. By virtue of their location in the vascular wall, ECs are exposed to the mechanical forces associated with blood flow. Fluid shear stress (flow) modulates vessel structure and function and is one of the most important hemodynamic forces.¹ Among many signal molecules that are activated by flow, members of the mitogen-activated protein kinase family, including extracellular signal–regulated kinase (ERK)1/2,^2–4 c-Jun NH₂-terminal kinase,⁵ and big mitogen–activated protein kinase/ERK5 are likely to be important for changes in EC gene expression.⁶

See page 1755

Our laboratory has investigated the early mechanical transduction events stimulated by flow. Results from our laboratory and others have shown that flow activated ERK1/2 in an integrin-dependent manner and that flow activated signal molecules that were localized to focal adhesions.^7,8 However, we observed that neither focal adhesion kinase (FAK) nor paxillin was rapidly phosphorylated by flow. Crk-associated substrate (Cas) is a potential adaptor protein for integrin-mediated cell adhesion and is phosphorylated by flow in a calcium-dependent manner.⁹ Cas is also associated with FAK and proline-rich tyrosine kinase (PYK2) through SH3 domain interactions. A cooperative interaction between PYK2 and c-Src has been shown to be required for tyrosine phosphorylation of Cas in Cos-7 cells.¹⁰ Therefore, we studied the role of PYK2 in flow signaling.

PYK2, also known as RAFTK/CAKß/CADTK/FAK2, is related to FAK and is regulated by several extracellular stimuli.^11–13 PYK2 is activated by hormones, G-protein–coupled receptor agonists, stress stimuli, membrane depolarization, and increasing intracellular calcium.^11,14 Activation of PYK2 has been implicated in the regulation of ion channels, ¹¹ cell adhesion and motility, ^10,15 the activity of ERK,^11,16 c-Jun NH₂-terminal kinase,¹⁴ p70 S6 kinase,¹⁷ and phosphatidylinositol 3-kinase/Akt pathways.¹⁸ It has been found that autophosphorylation of PYK2 on Tyr402 leads to the binding of the Src SH2 domain and subsequent Src activation and PYK2 phosphorylation.¹⁹ The activated Src bound to PYK2 may phosphorylate PYK2 at Tyr579, Tyr580, and Tyr881, which promotes Grb2 (an adaptor protein) binding to PYK2 and enhances PYK2 kinase activity.^20,21 PYK2 physically associates with cytoskeletal proteins, such as paxillin and Cas, and may increase their tyrosine phosphorylation in response to agonists.^11,19,22

Previously, our data have shown that c-Src is activated within 2 minutes of flow and that tyrosine phosphorylation of Cas requires calcium-dependent c-Src activation.⁹ Therefore, we hypothesized that flow-mediated activation of PYK2 phosphorylates Cas on the basis of several findings: (1) Flow induces intracellular calcium.²³ (2) PYK2 is a calcium-dependent tyrosine kinase.¹¹ (3) Flow activates Src and induces Cas phosphorylation, and PYK2 is reported to be a Cas tyrosine kinase.^9,10 In addition, because reactive oxygen species (ROS) production is induced by flow in ECs and because PYK2 is activated by ROS,^24,25 we examined the interaction between ROS and PYK2. In the present study, we found that PYK2 is an important and proximal target of flow-derived ROS and intracellular calcium in bovine aortic ECs (BAECs) and is also an upstream mediator of Cas in response to flow.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
A23187, phorbol 12-myristate 13-acetate (PMA), and N-acetyl-l-cysteine (NAC) were obtained from Sigma Chemical Co. BAPTA-AM, U73122, cytochalasin D (CD), and PP2 (an Src family kinase inhibitor) were purchased from Calbiochem. Monoclonal anti-PYK2 and p130 Cas antibodies were from Transduction Laboratories. Monoclonal anti-Src (clone GD11) antibody and anti-phosphotyrosine antibody (4G10) were purchased from Upstate Biotechnology. Anti-Myc antibody was from Invitrogen. Anti–hemagglutinin antigen (HA) tag antibodies (12CA5) were purchased from Roche Molecular Biochemicals. Protein A/G agarose beads were from Santa Cruz.

Cell Culture and Transfections
BAECs were harvested from fetal calf aortas by collagenase, as described previously.²⁶ Cells were grown in M199 (Life Technologies, Inc) supplemented with 10% FCS (Hyclone Laboratories) at 37°C in a 5% CO₂/95% air atmosphere. The wild-type (WT) CADTK/PYK2 and kinase-deficient CADTK/PYK2 cDNA constructs tagged with Myc peptide were kindly provided by Dr Li (University of North Carolina, Chapel Hill).¹⁴ The expression vector encoding p130^CAS was obtained from Dr Vuori (The Burnham Institute, La Jolla, Calif)²⁷ and was subcloned into another vector with the HA tag. Transient transfections were performed by using LipofectAmine (Invitrogen Life Technologies) according to the manufacturer’s directions.

Shear Stress Protocol
BAECs were grown to confluence on gelatin-coated dishes and were serum-starved for 3 to 6 hours. Before the experiment, cells were rinsed free of culture medium with HEPES-buffered saline solution (130 mmol/L NaCl, 5 mmol/L KCl, 1.5 mmol/L CaCl₂, 1.0 mmol/L MgCl₂, and 20 mmol/L HEPES, pH 7.4) plus 10 mmol/L glucose and were either maintained in static condition or exposed to flow in a cone-and-plate viscometer at 37°C, as described previously.²⁸

Immunoprecipitation and Western Blotting
After treatment, cells were washed with cold PBS and lysed in lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L Na₃VO₄, 1 mmol/L NaF, 2 mmol/L benzamidine, and 10 µg/mL leupeptin). The lysates were immunoprecipitated, and immune complexes were recovered by the addition of protein A/G agarose beads. The beads were washed 3 times with lysis buffer. For Western blot analysis, cell lysates or immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech), as described previously.²⁹ The membranes were blocked and incubated with primary antibody for 2 hours, followed by incubation for 1 hour with secondary antibody.

Adenoviral Transfection
BAECs were grown on 60-mm tissue culture dishes. On subconfluence, cells were incubated with M199 supplemented with 10% FCS containing Ad.KI-Src (where Ad indicates adenovirus and KI indicates kinase inactive) or Ad.LacZ for 2 days. The construction and preparation of Ad.KI-Src, the characterization of transfection efficiency, and control for cell toxicity have been described previously.⁹

Statistics and Densitometric Analysis
Data are presented as mean±SE. All experiments were performed at least 3 times. Significant differences were determined by the Student t test (P<0.05). Analysis of densitometry was performed by using NIH Image 1.60. Tyrosine phosphorylation was normalized first to the amount of protein on the basis of Western blot density. Results were then normalized for comparison among different experiments by arbitrarily setting the densitometric value of control cells or uninfected cells to 1.0.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Time-Dependent Phosphorylation of PYK2 by Shear Stress in BAECs
To find the kinase upstream from Cas stimulated by shear stress, we studied tyrosine phosphorylation of PYK2. BAECs were exposed to flow for varying times and harvested for analysis of PYK2 phosphorylation. PYK2 was immunoprecipitated, and the immunoprecipitated protein complexes were immunoblotted with 4G10, an anti-phosphotyrosine antibody. Tyrosine phosphorylation of PYK2 occurred as early as 0.5 minutes, peaked at 2 minutes (1.8±0.09-fold increase), and returned to near baseline by 40 minutes after stimulation (Figure 1A, top, and Figure 1B). There was no significant change in PYK2 protein level during these experiments (Figure 1A, bottom).

View larger version (26K): [in this window] [in a new window]   Figure 1. Time course for PYK2 tyrosine phosphorylation by shear stress. Serum-starved BAECs were either kept as static controls (represented by time 0) or were exposed to flow (shear stress 12 dyne/cm2) for the indicated times. A, Cell lysates were subjected to immunoprecipitation (IP) with anti-PYK2 antibody and analyzed by Western blot with anti-phosphotyrosine (YP) antibody (top). The bound antibodies were detected by enhanced chemiluminescence. To assess the reproducibility of PYK2 immunoprecipitation, the membrane was reprobed with anti-PYK2 antibody (bottom). B, Densitometric analysis of phosphorylated form of PYK2 in BAECs is shown. Results are mean±SE from 3 separate experiments.

Shear Stress–Induced PYK2 Phosphorylation Is Dependent on Intracellular Calcium and PLC Activity in BAECs
We and other groups have shown that shear stress stimulates a rapid increase in EC intracellular calcium that is dependent on the magnitude of shear stress and have found that flow-induced Cas phosphorylation is dependent on calcium.⁹ Increasing intracellular calcium by treatment of BAECs with 10 µmol/L A23187 (a calcium ionophore) for 5 minutes stimulated PYK2 phosphorylation (Figure 2). The magnitude of PYK2 phosphorylation in response to shear stress was similar to that observed with A23187 treatment (Figure 2A). We used BAPTA-AM to chelate intracellular calcium and EGTA to chelate extracellular calcium. Flow-induced PYK2 phosphorylation was inhibited by 30 µmol/L BAPTA-AM but not by 2 mmol/L EGTA alone (Figure 2B). These results indicate that increases in intracellular calcium are necessary for PYK2 activation by flow. Previous data had shown that shear stress stimulated phospholipase C (PLC) with increases in inositol trisphosphate formation and diacylglycerol and the subsequent mobilization of calcium from intracellular stores and activation of protein kinase C (PKC).³⁰ In the present study, U73122, an inhibitor of PLC, blocked the phosphorylation of PYK2 induced by flow (Figure 2C). These results indicate that PYK2 activation by flow is mediated through PLC, which is known to increase intracellular calcium. Next, we examined whether PKC activity is required for PYK2 phosphorylation by flow. PYK2 phosphorylation was significantly stimulated by treatment with 200 nmol/L PMA for 5 minutes (Figure 2D, lanes 7 and 8). To deplete PKC, BAECs were pretreated with PMA (1 µmol/L) for 20 hours before cells were exposed to flow or PMA. Long-term treatment with PMA inhibited PMA (200 nmol/L)–induced PYK2 activity (lanes 5 and 6) but did not inhibit flow-induced PYK2 phosphorylation (lanes 3 and 4). We also treated cells with 5 µmol/L chelerythrine chloride, a PKC inhibitor, for 30 minutes and found that the inhibitor could not block flow-mediated PYK2 phosphorylation (data not shown). These data show that PYK2 phosphorylation by flow requires PLC activation and mobilization of intracellular calcium, but not PKC activity.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of various inhibitors on flow-induced PYK2 phosphorylation. A, Serum-starved BAECs were exposed to flow (shear stress 12 dyne/cm2 for 2 minutes) or treated with A23187 (10 µmol/L for 5 minutes). B, Extracellular calcium was chelated by adding 2 mmol/L EGTA in Ca2+-free flow buffer for 30 minutes. To deplete intracellular calcium, BAECs were treated with 30 µmol/L BAPTA-AM for 30 minutes before exposure to flow (shear stress 24 dyne/cm2 for 2 minutes). C, Serum-starved BAECs were in the absence or presence of U73122 (5 µmol/L for 30 minutes) before they were exposed to flow (shear stress 24 dyne/cm2 for 2 minutes). D, To deplete PKC, BAECs were treated with PMA (1 µmol/L) for 20 hours before exposure to flow (shear stress 24 dyne/cm2 for 2 minutes) or PMA (200 nmol/L for 5 minutes). To stimulate PYK2 activity, BAECs were treated with PMA (200 nmol/L for 5 minutes). E, Serum-stared BAECs were in the absence or presence of CD (0.5 µmol/L for 30 minutes) before they were exposed to flow (shear stress 24 dyne/cm2 for 2 minutes). PYK2 phosphorylation and protein were analyzed as described above.

Effect of Cytoskeleton-Disrupting Agents on Flow-Induced PYK2 Phosphorylation
Recent data have shown that disruption of integrin clustering at focal adhesions prevents PYK2 phosphorylation in response to stimuli.³¹ To investigate a possible connection between PYK2 and the actin-based cytoskeleton, cells were pretreated with CD, a specific actin microfilament inhibitor, for 30 minutes before flow. As shown in Figure 2E, we observed that CD had no effect on flow-induced PYK2 phosphorylation, indicating that PYK2 phosphorylation does not require intact actin-based cytoskeleton organization.

Shear Stress–Induced PYK2 Phosphorylation Is Dependent on ROS
Previously, we and other groups have observed that shear stress increases ROS and that this leads to increased calcium.^23,24 Because PYK2 has been suggested to be a redox-sensitive kinase, we determined the effect of NAC on flow-mediated PYK2 activity. After preincubation of BAECs for 30 minutes with 30 mmol/L NAC, BAECs were exposed to flow or maintained in the static condition. As shown in Figure 3A, shear stress–induced PYK2 phosphorylation was inhibited significantly by NAC treatment (47±2.2%). We also examined whether NAC has the same effect on Cas phosphorylation induced by flow. We treated BAECs with 30 mmol/L NAC for 30 minutes, and then cells were exposed to flow for 10 minutes. Treatment with NAC dramatically inhibited Cas phosphorylation by flow (Figure 3B). These data suggest that phosphorylation of PYK2 and Cas are both regulated by ROS.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of NAC on PYK2 and Cas phosphorylation by shear stress. A, Serum-starved BAECs were pretreated for 30 minutes with 30 mmol/L NAC. Then cells were exposed to flow (shear stress 12 dyne/cm2 for 5 minutes). B, Cells were pretreated for 30 minutes with 30 mmol/L NAC. Then cells were exposed to flow (shear stress 12 dyne/cm2 for 10 minutes). Cell lysates were prepared, immunoprecipitated with anti-PYK2/Cas antibody, and analyzed by Western blot with either YP antibody or anti-PYK2/CAS antibody. Graphs at the bottom represent average data expression as fold increase in PYK2/Cas phosphorylation over that in unstimulated cells. Results are mean±SE from 3 separate experiments.

H₂O₂ Stimulates PYK2 Activity in BAECs, Which Was Inhibited by NAC
To evaluate the role of ROS in shear stress–induced PYK2 phosphorylation in BAECs, we used 2 approaches: (1) We tested the ability of exogenous ROS to induce phosphorylation of PYK2. (2) We assessed the effect of antioxidants on H₂O₂-induced PYK2 activation. We found that H₂O₂ time- and dose-dependently increased PYK2 phosphorylation. H₂O₂ (500 µmol/L) dramatically increased tyrosine phosphorylation of PYK2, with a peak at 5 minutes, and was sustained for 20 minutes (Figure 4A). H₂O₂ at a concentration of 100 µmol/L stimulated PYK2 phosphorylation, and 1 mmol/L H₂O₂ maximally stimulated PYK2 phosphorylation (Figure 4B). There was complete inhibition of H₂O₂-induced PYK2 phosphorylation at concentrations of 30 mmol/L NAC (Figure 4C), similar to results obtained with shear stress. Taken together, these results suggest that PYK2 is a redox-sensitive kinase and that H₂O₂ may mimic the effect of shear stress.

View larger version (38K): [in this window] [in a new window]   Figure 4. Activation of PYK2 by H2O2 was inhibited by pretreatment with NAC and calcium chelators. A, Serum-starved BAECs were stimulated with 500 µmol/L H2O2 for the indicated times. B, BAECs were treated with the indicated concentrations of H2O2 for 5 minutes. C, BAECs were pretreated for 30 minutes with 30 mmol/L NAC. Then cells were treated with 500 µm H2O2 for 5 minutes. D, BAECs were pretreated for 30 minutes with 30 µmol/L BAPTA-AM or 2 mmol/L EGTA in Ca2+-free flow buffer. Then cells were treated with 500 µm H2O2 for 5 minutes. PYK2 phosphorylation and protein were analyzed as described in Figure 1.

H₂O₂-Induced PYK2 Phosphorylation Is Calcium Dependent
To determine further the role of ROS in flow-mediated activation of PYK2, the calcium dependence of H₂O₂-induced PYK2 activation was examined. We observed that intracellular calcium chelation with 30 µmol/L BAPTA-AM for 30 minutes dramatically inhibited tyrosine phosphorylation of PYK2 by H₂O₂. However, EGTA, which chelates only extracellular calcium, had no effect (Figure 4D).

Effect of WT- and KI-PYK2 on Shear Stress–Induced Phosphorylation of Cas
To characterize further the role of PYK2 in shear stress–mediated phosphorylation of Cas, we cotransfected BAECs with HA-Cas and Myc-tagged WT-PYK2 or KI-PYK2 (Lys457 replaced by Ala), followed by exposure to flow for 5 minutes. The epitope-tagged Myc-PYK2 and HA-Cas were immunoprecipitated by anti-Myc and anti-HA antibodies, respectively. Immunoblots were then probed with anti-phosphotyrosine antibody. Two results were obtained: (1) We found that transfected Myc-WT-PYK2 was highly phosphorylated on tyrosine under basal conditions and was not further phosphorylated by flow (compare lanes 2 and 5 in Figure 5A). (2) KI-PYK2 was not phosphorylated basally and could not be stimulated by flow (Figure 5A; compare lanes 3 and 6), suggesting that autophosphorylation is essential for PYK2 activation by flow. As shown in Figure 5B, we observed that overexpression of WT-PYK2 dramatically increased HA-Cas phosphorylation basally (compare lanes 1 and 2), whereas KI-PYK2 had no effect (compare lanes 1 and 3). In cells transfected with WT-PYK2, flow-mediated tyrosine phosphorylation of HA-Cas was enhanced (compare lanes 4 and 5). Transfection of KI-PYK2 significantly inhibited flow-induced HA-Cas phosphorylation (compare lanes 5 and 6). Thus, KI-PYK2 acts as a dominant-negative kinase in the flow-mediated pathway. These results show that PYK2 is an upstream mediator of Cas tyrosine phosphorylation in response to flow.

View larger version (31K): [in this window] [in a new window]   Figure 5. The effect of WT- and KI-PYK2 on shear stress–induced phosphorylation of Cas. Epitope-tagged HA-Cas (1 µg) was cotransfected with 1 µg pcDNA 3.1 (Invitrogen) empty vector or epitope-tagged Myc of WT-PYK2 or KI-PYK2 into BAECs. The transfected cells were serum-starved before they were subjected to flow (shear stress 12 dyne/cm2 for 5 minutes). Cell lysates were immunoprecipitated with anti-Myc (A) or anti-HA (B) antibody and analyzed by Western blot with anti-YP, PYK2, or Cas antibody.

Shear Stress–Induced Tyrosine Phosphorylation of PYK2 Is Not Dependent on Src Kinase
c-Src has been shown to be activated rapidly by flow in BAECs, and flow-induced Cas phosphorylation requires c-Src activity.⁹ Because we found that PYK2 is a Cas kinase stimulated by flow (Figure 5B), we studied the role of c-Src activity in flow-induced PYK2 phosphorylation. We pretreated BAECs with 10 µmol/L PP2 for 30 minutes. PP2 inhibited the basal phosphorylation of PYK2 (Figure 6A; compare lanes 1 and 3 [80±3.5%]). In contrast, flow-induced PYK2 phosphorylation was not inhibited when normalized to the baseline (2.0±0.2- and 2.5±0.49-fold increases; Figure 6A, lanes 2 and 4). We next tested the effect of overexpression by adenovirus of KI-Src on PYK2 activity. We found that infection of BAECs with KI-Src (multiplicity of infection 1000) inhibited flow-induced Cas phosphorylation (55% inhibition, Figure 6B; compare lanes 4 and 6) but did not inhibit tyrosine phosphorylation of PYK2 by flow (Control: 2.1±0.14, LacZ: 2.5±0.21, KI-Src: 2.8±0.23; Figure 6C). We also observed that overexpression of KI-Src had no effect on H₂O₂-induced PYK2 phosphorylation (data not shown). Taken together, these data suggest that c-Src kinase activity is not involved in flow-induced PYK2 phosphorylation in BAECs.

View larger version (20K): [in this window] [in a new window]   Figure 6. Shear stress–induced PYK2 phosphorylation was not inhibited by KI-Src. A, Serum-starved BAECs were pretreated with 10 µmol/L PP2 for 30 minutes, followed by exposure to flow (shear stress 24 dyne/cm2 for 2 minutes, n=4) B and C, BAECs were infected with adenoviral containing KI-Src or Ad.Lac Z (Lac Z) for 48 hours. The infected cells were exposed to flow (shear stress 12 dyne/cm2 for 5 minutes). Cas/PYK2 phosphorylation and protein were analyzed as described in Figure 1 (n=1 in panel B and n=4 in panel C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of the present study are identification of a redox-sensitive pathway for flow-mediated activation PYK2 that requires ROS and intracellular calcium, but not Src kinase. Specifically, we found that (1) flow rapidly stimulated PYK2 tyrosine phosphorylation through ROS and intracellular calcium; (2) flow-induced PYK2 phosphorylation required activity of tyrosine kinases and PLC, but not c-Src; and (3) PYK2 is an upstream kinase for Cas.

We anticipated that PYK2 activation by flow would be mediated by ROS in ECs because flow increased ROS production in ECs, because flow-induced PYK2 phosphorylation was inhibited by NAC, and because H₂O₂ stimulated PYK2 activity. Previous studies have shown that ECs generate significant amounts of superoxide and hydrogen peroxide in response to stimuli that increase calcium and PKC activity.^32,33 Several groups have demonstrated that mechanical forces, such as stretch, oscillatory flow, and steady laminar shear stress, stimulate ROS production in cultured ECs.^24,34–36 We found that H₂O₂ and flow greatly induced PYK2 phosphorylation (Figures 1A and 4A), which was inhibited by NAC, a well-known precursor of glutathione synthesis (Figures 3A and 4B). Our findings are in good agreement with the observations that H₂O₂ activates PYK2 in cultured vascular smooth muscle cells and that PYK2 can be activated by other ROS generators, such as angiotensin II, platelet-derived growth factor, cytokines, and ultraviolet radiation.^25,37–40 The mechanism by which ROS are generated in ECs exposed to flow remains unclear but may involve small G-protein–mediated activation of gp91-phox.^41,42

PYK2 phosphorylation is calcium dependent in response to various stimuli. There are several pathways to increase calcium concentration. Shear stress activated a pertussis toxin–sensitive G-protein–coupled K⁺ channel, which changes membrane potential. The resulting hyperpolarization, via effects on voltage-sensitive cation channels, may increase calcium influx. Other groups have shown that shear stress activates PLC to form inositol trisphosphate and increase calcium concentration from intracellular stores.³⁰ Chen et al⁸ have reported that receptor tyrosine kinases, such as flk-1 and integrins, can serve as mechanosensors in ECs in response to flow. PLC has been shown to be recruited to flk-1. Presumably, flow induced the phosphorylation flk-1, which then associated with SH2-containing proteins, such as PLC, increasing intracellular calcium. Recently, we observed that PLC was phosphorylated in response to shear stress (data not shown). We found that flow-induced PYK2 phosphorylation was inhibited by BAPTA-AM, but not by EGTA (Figure 2B), indicating that intracellular calcium is more important than extracellular calcium in flow activation of PYK2. It is possible that a receptor tyrosine kinase is involved in this pathway. The role of flk-1 in flow-induced PYK2 activation will be examined in the future.

The role of Src family kinases in PYK2 phosphorylation is complex. We found that PP2, a Src family tyrosine kinase inhibitor, did not inhibit flow-induced PYK2 phosphorylation but did inhibit the basal level of PYK2 phosphorylation. Overexpression of KI-Src inhibited flow-induced Cas phosphorylation in BAECs (Figure 6B) and human umbilical vein endothelial cells⁹ but had no effect on flow-induced PYK2 activity (Figure 6C). Thus, we believe that Src is likely involved in PYK2 phosphorylation basally but not in response to flow. However, other groups observed that lysophosphatidic acid–induced activation of PYK2 was normal in Src-/- fibroblasts but decreased in Src-/- Yes-/- Fyn-/- fibroblasts in response to lysophosphatidic acid, bradykinin, carbachol, and angiotensin II.⁴³ It is likely that different members of the Src family kinase bind to PYK2 and phosphorylate PYK2 in response to different stimuli in various cell types. Alternatively, a tyrosine kinase other than Src family kinase may be involved, as suggested by the failure of PP2 to block the flow-mediated increase in PYK2 phosphorylation.

We speculate that integrin-mediated events are important in shear stress–induced signal transduction, because in response to flow, paxillin changes its alignment and FAK is phosphorylated.^44,45 We found that PYK2 activation was not attenuated by disruption with CD (Figure 2E), suggesting that PYK2 activation in response to flow is via a signaling pathway separate from actin microfilament integrity. This observation is similar to our previous findings showing that actin microfilament integrity is not required for flow-induced big mitogen–activated protein kinase and ERK1/2 activity.⁷

In summary, the present study is the first to demonstrate that shear stress stimulates PYK2 tyrosine phosphorylation through ROS and intracellular calcium in ECs. Cas is a downstream target of PYK2 in response to flow in ECs. This shear stress–induced PYK2 pathway is important in EC function related to focal adhesions, such as migration and cell shape change, inasmuch as PYK2 and Cas are colocalized to focal adhesions.

	Footnotes

 
*These authors contributed equally to the present study.

Received June 19, 2002; accepted August 6, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995; 75: 519–560.[Abstract/Free Full Text]

2. Hsieh H-J, Li N-Q, Frangos JA. Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J Cell Physiol. 1992; 150: 552–558.[CrossRef][Medline] [Order article via Infotrieve]

3. Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci U S A. 1994; 91: 4678–4682.[Abstract/Free Full Text]

4. Schwachtgen JL, Houston P, Campbell C, Sukhatme V, Braddock M. Fluid shear stress activation of egr-1 transcription in cultured human endothelial and epithelial cells is mediated via the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway. J Clin Invest. 1998; 101: 2540–2549.[Medline] [Order article via Infotrieve]

5. Li YS, Shyy JY, Li S, Lee J, Su B, Karin M, Chien S. The Ras-JNK pathway is involved in shear-induced gene expression. Mol Cell Biol. 1996; 16: 5947–5954.[Abstract]

6. Yan C, Takahashi M, Okuda M, Lee JD, Berk BC. Fluid shear stress stimulates big mitogen-activated protein kinase 1 (BMK1) activity in endothelial cells: dependence on tyrosine kinases and intracellular calcium. J Biol Chem. 1999; 274: 143–150.[Abstract/Free Full Text]

7. Takahashi M, Berk BC. Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells: essential role for a herbimycin-sensitive kinase. J Clin Invest. 1996; 98: 2623–2631.[Medline] [Order article via Infotrieve]

8. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, Shyy JY. Mechanotransduction in response to shear stress: roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem. 1999; 274: 18393–18400.[Abstract/Free Full Text]

9. Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999; 274: 26803–26809.[Abstract/Free Full Text]

10. Astier A, Manie SN, Avraham H, Hirai H, Law SF, Zhang Y, Golemis EA, Fu Y, Druker BJ, Haghayeghi N, Freedman AS, Avraham S. The related adhesion focal tyrosine kinase differentially phosphorylates p130Cas and the Cas-like protein, p105HEF1. J Biol Chem. 1997; 32: 19719–19724.

11. Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995; 376: 737–745.[CrossRef][Medline] [Order article via Infotrieve]

12. Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE, et al. Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem. 1995; 270: 27742–27751.[Abstract/Free Full Text]

13. Schaller MD, Sasaki T. Differential signaling by the focal adhesion kinase and cell adhesion kinase beta. J Biol Chem. 1997; 272: 25319–25325.[Abstract/Free Full Text]

14. Yu H, Li X, Marchetto GS, Dy R, Hunter D, Calvo B, Dawson TL, Wilm M, Anderegg RJ, Graves LM, Earp HS. Activation of a novel calcium-dependent protein-tyrosine kinase. J Biol Chem. 1996; 271: 29993–29998.[Abstract/Free Full Text]

15. Lakkakorpi P, Nakamura I, Nagy R, Parsons J, Rodan G, Duong L. Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone. J Biol Chem. 1999; 274: 4900–4907.[Abstract/Free Full Text]

16. Murasawa S, Mori Y, Nozawa Y, Masaki H, Maruyama K, Tsutsumi Y, Moriguchi Y, Shibasaki Y, Tanaka Y, Iwasaka T, Inada M, Matsubara H. Role of calcium-sensitive tyrosine kinase Pyk2/CAKbeta/RAFTK in angiotensin II induced Ras/ERK signaling. Hypertension. 1998; 32: 668–675.[Abstract/Free Full Text]

17. Graves LM, He Y, Lambert J, Hunter D, Li X, Earp HS. An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells. J Biol Chem. 1997; 272: 1920–1928.[Abstract/Free Full Text]

18. Rocic P, Lucchesi PA. Down-regulation by antisense oligonucleotides establishes a role for the proline-rich tyrosine kinase PYK2 in angiotensin II-induced signaling in vascular smooth muscle. J Biol Chem. 2001; 276: 21902–21906.[Abstract/Free Full Text]

19. Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J. A role for Pyk2 and src in linking G-protein-coupled receptors with MAP kinase activation. Nature. 1996; 383: 547–550.[CrossRef][Medline] [Order article via Infotrieve]

20. Blaukat A, Ivankovic-Dikic I, Gronroos E, Dolfi F, Tokiwa G, Vuori K, Dikic I. Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades. J Biol Chem. 1999; 274: 14893–14901.[Abstract/Free Full Text]

21. Li X, Dy RC, Cance WG, Graves LM, Earp HS. Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase. J Biol Chem. 1999; 274: 8917–8924.[Abstract/Free Full Text]

22. Ohba T, Ishino M, Aoto H, Sasaki T. Interaction of two proline-rich sequences of cell adhesion kinase beta with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf. Biochem J. 1998; 330: 1249–1254.[Medline] [Order article via Infotrieve]

23. Ishida T, Takahashi M, Corson MA, Berk BC. Fluid shear stress-mediated signal transduction: How do endothelial cells transduce mechanical force into biological responses? Ann N Y Acad Sci. 1997; 811: 12–24.[Medline] [Order article via Infotrieve]

24. Laurindo FR, Pedro MdA, Barbeiro HV, Pileggi F, Carvalho MH, Augusto O, da Luz PL. Vascular free radical release: ex vivo and in vivo evidence for a flow-dependent endothelial mechanism. Circ Res. 1994; 74: 700–709.[Abstract/Free Full Text]

25. Frank GD, Motley ED, Inagami T, Eguchi S. PYK2/CAKbeta represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells. Biochem Biophys Res Commun. 2000; 270: 761–765.[CrossRef][Medline] [Order article via Infotrieve]

26. Gimbrone MA Jr. Culture of vascular endothelium. Prog Hemost Thromb. 1976; 3: 1–28.[Medline] [Order article via Infotrieve]

27. Vuori K, Hirai H, Aizawa S, Ruoslahti E. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol Cell Biol. 1996; 16: 2606–2613.[Abstract]

28. Traub O, Yan C, Berk BC. In vitro stimulation of shear stress and mitogen-activated protein kinase responses to shear stress in endothelial cells.In: Lelkes P, ed. Mechanical Forces and the Endothelium. Amsterdam, Netherlands: Hardwood Academic Publishers; 1998: 89–109.

29. Tseng H, Peterson TE, Berk BC. Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. Circ Res. 1995; 77: 869–878.[Abstract/Free Full Text]

30. Berk BC, Corson MA, Peterson TE, Tseng H. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech. 1995; 28: 1439–1450.[CrossRef][Medline] [Order article via Infotrieve]

31. Park SY, Avraham H, Avraham S. Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation. J Biol Chem. 2000; 275: 19768–19777.[Abstract/Free Full Text]

32. Matsubara T, Ziff M. Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore. J Cell Physiol. 1986; 127: 207–210.[CrossRef][Medline] [Order article via Infotrieve]

33. Royall JA, Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H₂O₂ in cultured endothelial cells. Arch Biochem Biophys. 1993; 302: 348–355.[CrossRef][Medline] [Order article via Infotrieve]

34. Fleming I, Bauersachs J, Fisslthaler B, Busse R. Ca²⁺-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res. 1998; 82: 686–695.[Abstract/Free Full Text]

35. De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox-state: role of a superoxide-producing NADH oxidase. Circ Res. 1998; 82: 1094–1101.[Abstract/Free Full Text]

36. Howard AB, Alexander RW, Nerem RM, Griendling KK, Taylor WR. Cyclic strain induces an oxidative stress in endothelial cells. Am J Physiol. 1997; 272: C421–C427.[Medline] [Order article via Infotrieve]

37. Tokiwa G, Dikic I, Lev S, Schlessinger J. Activation of Pyk2 by stress signals and coupling with JNK signaling pathway. Science. 1996; 273: 792–794.[Abstract]

38. Sabri A, Govindarajan G, Griffin TM, Byron KL, Samarel AM, Lucchesi PA. Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle. Circ Res. 1998; 83: 841–851.[Abstract/Free Full Text]

39. Brinson AE, Harding T, Diliberto PA, He Y, Li X, Hunter D, Herman B, Earp HS, Graves LM. Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. J Biol Chem. 1998; 273: 1711–1718.[Abstract/Free Full Text]

40. Eguchi S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa T, Motley ED, Owada KM, Marumo F, Hirata Y. Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension. 1999; 33: 201–206.[Abstract/Free Full Text]

41. Hiraoka W, Vazquez N, Nieves-Neira W, Chanock SJ, Pommier Y. Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells. J Clin Invest. 1998; 102: 1961–1968.[Medline] [Order article via Infotrieve]

42. He L, Chen J, Dinger B, Sanders K, Sundar K, Hoidal J, Fidone S. Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice. Am J Physiol. 2002; 282: C27–C33.

43. Andreev J, Galisteo ML, Kranenburg O, Logan SK, Chiu ES, Okigaki M, Cary LA, Moolenaar WH, Schlessinger J. Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade. J Biol Chem. 2001; 276: 20130–20135.[Abstract/Free Full Text]

44. Girard PR, Nerem RM. Endothelial cell signaling and cytoskeletal changes in response to shear stress. Front Med Biol Eng. 1993; 5: 31–36.[Medline] [Order article via Infotrieve]

45. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY. Fluid shear stress activation of focal adhesion kinase: linking to mitogen-activated protein kinases. J Biol Chem. 1997; 272: 30455–30462.[Abstract/Free Full Text]

This article has been cited by other articles:

				J.-L. Balligand, O. Feron, and C. Dessy eNOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues Physiol Rev, April 1, 2009; 89(2): 481 - 534. [Abstract] [Full Text] [PDF]

				G. Osol and M. Mandala Maternal Uterine Vascular Remodeling During Pregnancy Physiology, February 1, 2009; 24(1): 58 - 71. [Abstract] [Full Text] [PDF]

				W.-Z. Ying, K. Aaron, and P. W. Sanders Dietary Salt Activates an Endothelial Proline-Rich Tyrosine Kinase 2/c-Src/Phosphatidylinositol 3-Kinase Complex to Promote Endothelial Nitric Oxide Synthase Phosphorylation Hypertension, December 1, 2008; 52(6): 1134 - 1141. [Abstract] [Full Text] [PDF]

				W.-Z. Ying, K. Aaron, and P. W. Sanders Mechanism of dietary salt-mediated increase in intravascular production of TGF-{beta}1 Am J Physiol Renal Physiol, August 1, 2008; 295(2): F406 - F414. [Abstract] [Full Text] [PDF]

				J. O. Fledderus, R. A. Boon, O. L. Volger, H. Hurttila, S. Yla-Herttuala, H. Pannekoek, A.-L. Levonen, and A. J.G. Horrevoets KLF2 Primes the Antioxidant Transcription Factor Nrf2 for Activation in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., July 1, 2008; 28(7): 1339 - 1346. [Abstract] [Full Text] [PDF]

				B. Fisslthaler, A. E. Loot, A. Mohamed, R. Busse, and I. Fleming Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin Circ. Res., June 20, 2008; 102(12): 1520 - 1528. [Abstract] [Full Text] [PDF]

				M. J. Allingham, J. D. van Buul, and K. Burridge ICAM-1-Mediated, Src- and Pyk2-Dependent Vascular Endothelial Cadherin Tyrosine Phosphorylation Is Required for Leukocyte Transendothelial Migration J. Immunol., September 15, 2007; 179(6): 4053 - 4064. [Abstract] [Full Text] [PDF]

				L. I. Plotkin, S. C. Manolagas, and T. Bellido Glucocorticoids Induce Osteocyte Apoptosis by Blocking Focal Adhesion Kinase-mediated Survival: EVIDENCE FOR INSIDE-OUT SIGNALING LEADING TO ANOIKIS J. Biol. Chem., August 17, 2007; 282(33): 24120 - 24130. [Abstract] [Full Text] [PDF]

				J. Seebach, G. Donnert, R. Kronstein, S. Werth, B. Wojciak-Stothard, D. Falzarano, C. Mrowietz, S. W. Hell, and H.-J. Schnittler Regulation of endothelial barrier function during flow-induced conversion to an arterial phenotype Cardiovasc Res, August 1, 2007; 75(3): 598 - 607. [Abstract] [Full Text] [PDF]

				J. Guo, A. Sabri, H. Elouardighi, V. Rybin, and S. F. Steinberg {alpha}1-Adrenergic Receptors Activate AKT via a Pyk2/PDK-1 Pathway That Is Tonically Inhibited by Novel Protein Kinase C Isoforms in Cardiomyocytes Circ. Res., December 8, 2006; 99(12): 1367 - 1375. [Abstract] [Full Text] [PDF]

				S. Lehoux Redox signalling in vascular responses to shear and stretch Cardiovasc Res, July 15, 2006; 71(2): 269 - 279. [Abstract] [Full Text] [PDF]

				D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]

				R. F. Wu, Y. C. Xu, Z. Ma, F. E. Nwariaku, G. A. Sarosi Jr., and L. S. Terada Subcellular targeting of oxidants during endothelial cell migration J. Cell Biol., December 5, 2005; 171(5): 893 - 904. [Abstract] [Full Text] [PDF]

				R. Koshida, P. Rocic, S. Saito, T. Kiyooka, C. Zhang, and W. M. Chilian Role of Focal Adhesion Kinase in Flow-Induced Dilation of Coronary Arterioles Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2548 - 2553. [Abstract] [Full Text] [PDF]

				R. Magid and P. F. Davies Endothelial Protein Kinase C Isoform Identity and Differential Activity of PKC{zeta} in an Athero-Susceptible Region of Porcine Aorta Circ. Res., September 2, 2005; 97(5): 443 - 449. [Abstract] [Full Text] [PDF]

				L.-k. Tai, Q. Zheng, S. Pan, Z.-G. Jin, and B. C. Berk Flow Activates ERK1/2 and Endothelial Nitric Oxide Synthase via a Pathway Involving PECAM1, SHP2, and Tie2 J. Biol. Chem., August 19, 2005; 280(33): 29620 - 29624. [Abstract] [Full Text] [PDF]

				J. D. van Buul, E. C. Anthony, M. Fernandez-Borja, K. Burridge, and P. L. Hordijk Proline-rich Tyrosine Kinase 2 (Pyk2) Mediates Vascular Endothelial-Cadherin-based Cell-Cell Adhesion by Regulating {beta}-Catenin Tyrosine Phosphorylation J. Biol. Chem., June 3, 2005; 280(22): 21129 - 21136. [Abstract] [Full Text] [PDF]

				Z.-G. Jin, C. Wong, J. Wu, and B. C. Berk Flow Shear Stress Stimulates Gab1 Tyrosine Phosphorylation to Mediate Protein Kinase B and Endothelial Nitric-oxide Synthase Activation in Endothelial Cells J. Biol. Chem., April 1, 2005; 280(13): 12305 - 12309. [Abstract] [Full Text] [PDF]

				R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]

				R. S. Frey and A. B. Malik Oxidant signaling in lung cells Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L1 - L3. [Full Text] [PDF]

				N. Lerner-Marmarosh, M. Yoshizumi, W. Che, J. Surapisitchat, H. Kawakatsu, M. Akaike, B. Ding, Q. Huang, C. Yan, B. C. Berk, et al. Inhibition of Tumor Necrosis Factor-{alpha}-Induced SHP-2 Phosphatase Activity by Shear Stress: A Mechanism to Reduce Endothelial Inflammation Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1775 - 1781. [Abstract] [Full Text] [PDF]

				Z.-G. Jin, H. Ueba, T. Tanimoto, A. O. Lungu, M. D. Frame, and B. C. Berk Ligand-Independent Activation of Vascular Endothelial Growth Factor Receptor 2 by Fluid Shear Stress Regulates Activation of Endothelial Nitric Oxide Synthase Circ. Res., August 22, 2003; 93(4): 354 - 363. [Abstract] [Full Text] [PDF]

				P. F. Davies Multiple Signaling Pathways in Flow-Mediated Endothelial Mechanotransduction: PYK-ing the Right Location Arterioscler. Thromb. Vasc. Biol., November 1, 2002; 22(11): 1755 - 1757. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 22/11/1790    most recent 01.ATV.0000034475.40227.40v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tai, L.-K. Articles by Berk, B. C. Search for Related Content PubMed PubMed Citation Articles by Tai, L.-K. Articles by Berk, B. C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLCYSTEINE CALCIUM COMPOUNDS CALCIUM, ELEMENTAL CYTOCHALASIN D HYDROGEN PEROXIDE Related Collections Cell signalling/signal transduction Endothelium/vascular type/nitric oxide