Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California at San Diego, La Jolla, California 92093
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ABSTRACT |
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Blood flow can modulate vascular cell
functions. We studied interactions between integrins and Flk-1 in
transducing the mechanical shear stress due to flow. This application
of a step shear stress caused Flk-1 · Casitas B-lineage
lymphoma (Cbl) activation (Flk-1 · Cbl association, tyrosine
phosphorylation of the Cbl-bound Flk-1, and tyrosine phosphorylation of
Cbl) in bovine aortic endothelial cells (BAECs). The activation of
integrins by plating BAECs on vitronectin or fibronectin also induced
this Flk-1 · Cbl activation. The shear-induced
Flk-1 · Cbl activation was blocked by inhibitory antibodies for
v
3- or
1-integrin,
suggesting that it is mediated by integrins. Inhibition of Flk-1 by
SU1498 also abolished this shear-induced Flk-1 · Cbl
activation. In contrast to the requirement of integrins for
Flk-1 · Cbl activation, the Flk-1 blocker SU1498 had no
detectable effect on the shear-induced integrin activation, suggesting
that integrins and Flk-1 play sequential roles in the signal
transduction hierarchy induced by shear stress. Integrins are essential
for the mechanical activation of Flk-1 by shear stress but not for the
chemical activation of Flk-1 by VEGF.
mechanotransduction; vascular endothelial growth factor; Casitas B-lineage lymphoma
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INTRODUCTION |
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MECHANICAL FORCES ARE KNOWN
TO PLAY A KEY ROLE in many physiological processes, but how cells
perceive mechanical stimuli and convert them into biochemical signaling
pathways is not yet well understood. Shear stress, the tangential
component of hemodynamic forces acting on the vessel wall, is an
important modulator of vascular cell functions in normal and
pathophysiological conditions, e.g., endothelial wound healing,
atherosclerosis, and reperfusion injury (14, 36, 37, 43).
In vitro experiments using endothelial cells (ECs) cultured in flow
channels have shown that the structure and function of ECs are
modulated by shear stress (6). Recent studies indicate
that the mechanochemical transduction in response to shear stress
involves different types of receptors and multiple intracellular
signaling pathways. Studies from several laboratories, including our
own, have demonstrated that shear stress can activate integrins, VEGF
receptor-2 (Flk-1), G protein-coupled receptors (GPCR), and ion
channels and subsequently regulate mitogen-activated protein kinases
and the NF-B pathway through Ras and Rho family GTPases (5,
26, 33, 40). The available evidence indicates that the shear
stress-induced signaling from different sensing elements can coordinate
downstream signaling through convergent and divergent pathways.
However, the possible interaction of these sensing elements in ECs in
response to shear stress remains to be investigated.
Most intracellular signals are not transduced along a simple linear
path involving the interaction of only one molecule with the next.
Proteins associate into networks, which are subject to control by many
interdependent processes. Many different types of receptors exert
mutual influences on each other (9, 16, 23, 30). There is
considerable evidence for the synergistic regulation of signals by
integrins and receptor tyrosine kinases (RTKs). Activation of integrins
by plating cells on ECM proteins leads to ligand-independent activation
of epidermal growth factor receptor (EGFR) in human dermal fibroblasts
and platelet-derived growth factor receptor (PDGFR) in human diploid
foreskin AG 1518 fibroblasts (24, 39) and enhances the
growth factor-induced tyrosine phosphorylation of PDGFR, EGFR, and
Flk-1 when compared with cells in suspension (22, 35).
Ligand activation of PDGFR (with PDGF) or Flk-1 (with VEGF) has also
been reported to induce the association of PDGFR or Flk-1 with
integrin, respectively (29, 35). Borges et al.
(2) have found that recombinant Flk-1 constitutively forms
a physical complex with integrin v
3 in
CHO cells. Given that shear stress can activate both integrins (12) and Flk-1 (4), it is of interest to
determine the interplay between integrins and RTKs in response to shear stress.
The activation of Flk-1 results in multiple molecular events, including the binding and phosphorylation of adapter proteins and the activation of MAPKs (13). Casitas B-lineage lymphoma (Cbl), an adapter protein, plays a central role in tyrosine kinase-related signal transduction (21, 34). Cbl phosphorylation and its association with other proteins are stimulated in cells after treatment with growth factors such as EGF (17, 42), PDGF (1), and colony-stimulating factor (CSF) (10). There is ample evidence that Cbl associates with RTKs (e.g., EGFR, PDGFR, and CSF-1R) and other adapter proteins (e.g., Grb2, Shc, and P85) to activate downstream signaling pathways (21). Our previous study indicates that shear stress induces the tyrosine phosphorylation of Cbl and its translocation from cytosol to the membrane (19). It is of interest to know whether Flk-1 binds to Cbl upon shear stress application and how the shear-modulated Flk-1 interacts with integrins.
In the current study, we investigated the interaction of integrins and Flk-1 in response to shear stress. We have shown that the association between Flk-1 and Cbl can be induced by shear stress and also by the activation of integrins through matrix binding. In response to shear stress, integrins regulate the activation of Flk-1 but not vice versa. Although both mechanical stimulus and chemical ligand can induce Flk-1 · Cbl activation and the downstream events, they differ in that integrins are essential for the action of shear stress but not for that of VEGF.
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EXPERIMENTAL PROCEDURES |
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Cell culture. Bovine aortic endothelial cells (BAECs) before passage 10 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM sodium pyruvate.
Cell adhesion assays. Confluent BAECs were serum starved for 12 h before detachment with 0.05% trypsin/0.33 mM EDTA. The cells were pelleted and resuspended in DMEM on 1% agarose gel for 60 min at 37°C. The cells in suspension were then seeded on six-well plates coated with vitronectin (0.2 µg/cm2) or fibronectin (2 µg/cm2) for various periods of time.
Shear stress experiments. A flow system (8) was used to impose shear stress on cultured ECs. In brief, a 75 × 38-mm glass slide seeded with a confluent monolayer of BAECs formed the floor of the flow chamber (0.025 cm in height, 2.5 cm in width, and 5.0 cm in length), which was created by sandwiching a silicone gasket between the glass slide and an acrylic plate. The cells were exposed to a step shear stress resulting from rapidly opening a valve between two reservoirs with different fluid heights. The pressure difference between the two reservoirs causes a plane Poiseuille flow in the parallel-plate flow chamber, leading to a laminar shear stress acting on cultured ECs. With the use of a flow rate at 30 ml/min, the shear stress generated in the flow chamber with the geometry mentioned above was controlled at 12 dyn/cm2. The circulating medium was kept at 37°C with 95% humidified air and 5% CO2.
Immunoprecipitation and immunoblotting.
The antibodies used for immunoprecipitation and immunoblotting
were polyclonal anti-Cbl, anti-Flk-1, anti-HA, and anti-ERK and
monoclonal anti-Myc, anti-phosphotyrosine (PY20; Santa Cruz Biotechnology), anti-v
3 (LM609),
anti-
1 (6S6; Chemicon), anti-phospho-ERK (Cell
Signaling), and anti-Shc (Transduction Laboratories).
Immunoprecipitation and immunoblotting were conducted as previously
described (15).
Immunostaining and fluorescence microscopy.
Integrin clustering was investigated by immunostaining. Cells
were fixed in methanol at 20°C for 2 min and incubated with the
anti-
v
3 LM609 mAb or the
anti-
1 6S6 mAb for 2 h at 37°C, followed by
incubation with FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch)
for 1 h at room temperature. Hoechst-33258 (Molecular Probes) was
used to stain the nucleus. The immunostaining was observed with a
confocal microscopy system (MRC-1000, Bio-Rad). The images were
analyzed by using NIH Image and Microsoft Excel software.
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RESULTS |
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Shear stress and VEGF induce Flk-1 · Cbl activation.
Cbl has been reported to associate with RTKs, including EGFR and PDGFR
(34). Because shear stress causes the phosphorylation of
Flk-1 (4), we investigated whether shear stress can induce the association of Flk-1 and Cbl. Lysates of sheared and control cells
were immunoprecipitated with an anti-Cbl antibody, followed by
immunoblotting with anti-Flk-1 and anti-PY20 antibodies. As shown in
Fig. 1 (top left), the
association between Flk-1 and Cbl showed a rapid and transient increase
after shearing. These changes became detectable at 1 min, peaked at 5 min, and returned to static control level by 15 min. By stripping the
nitrocellulose membrane and reprobing with an anti-PY20 antibody, we
observed a similar time course of the tyrosine-phosphorylation of Flk-1 associated with Cbl (Fig. 1, left center).
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Integrin-ligand interaction induces Flk · Cbl activation.
Integrin-ligand interaction has been reported to activate a variety of
RTKs, including EGFR and PDGFR (24, 39). We examined whether the engagement of integrins by their ligands is sufficient to
induce Flk-1 · Cbl activation. Cellular interaction with ECM proteins coated on solid surface can induce integrin activation and
subsequent signaling events (7). Therefore, we used this approach to activate integrins and assess their effect on
Flk-1 · Cbl activation. BAECs were plated on vitronectin
(ligand for v
3) or fibronectin (ligand
for both
5
1 and
v
3). Plating cells on vitronectin (Fig.
2, left) caused a significant
increase of Flk-1 · Cbl activation at 60 and 120 min. Plating
of BAECs on fibronectin caused an earlier and more transient increase
in Flk-1 · Cbl activation, which was noticeable at 30 min and
reached a peak at 60 min (Fig. 2, right). Beads coated with
anti-
1 or
v
3 antibodies
also induced a significant Flk-1 · Cbl activation at 20 min
(data not shown). These results indicate that integrin activation is
sufficient to cause Flk-1 · Cbl activation.
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Integrin integrity is essential for the Flk-1 · Cbl
activation induced by shear stress.
Shear stress can activate both integrins (12) and Flk-1
(4). Because integrins were proposed as potential
mechanosensors (12, 41), we examined whether integrins are
essential for the shear-induced Flk-1 · Cbl activation. We
preincubated BAECs with LM609 or 6S6, which are inhibitory antibodies
of v
3- and
1-integrins,
respectively, or with mouse IgG as a control. The pretreated cells were
subjected to shear stress for 5 min or kept as static control. Both
LM609 and 6S6 blocked the Cbl-bound Flk-1 phosphorylation and
Flk-1 · Cbl association induced by shear stress (Fig.
3). In contrast, noninhibitory antiserum
against
3- or
1-integrins had no
significant effect on the shear-induced Flk-1 · Cbl
activation (data not shown). Pretreating BAECs with LM609 or 6S6
also blocked the shear-induced tyrosine-phosphorylation of Cbl (Fig.
4), which provides docking sites for
multiple downstream signaling molecules (21). Our findings
indicate that the shear-induced Flk-1 · Cbl activation is
mediated by integrins.
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Flk-1 is important for Flk-1 · Cbl association and tyrosine
phosphorylation of Cbl.
We studied the role of Flk-1 in the shear activation of
Flk-1 · Cbl by using SU1498, which is a specific Flk-1
inhibitor (IC50 = 700 nM) that inhibits the
VEGF-induced Flk-1 autophosphorylation and DNA synthesis (20, 32,
38). SU1498 (5 µM) blocked the shear-induced
Flk-1 · Cbl association (Fig.
5A) and Cbl tyrosine phosphorylation (Fig. 5B), suggesting that a functional
Flk-1 is essential for the shear-induced Flk-1 · Cbl
association and subsequent Cbl phosphorylation.
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Flk-1 is not essential for the integrin-Shc association and
integrin clustering induced by shear stress.
Because integrin 3 has been shown to associate with
Flk-1 (2, 35), we studied whether integrins and Flk-1 are
parallel or serial in the signaling transduction machinery activated by shear stress. Having shown that integrins play a critical role in the
shear activation of Flk-1, we proceeded to examine the role of Flk-1 in
the shear activation of integrins. BAECs were preincubated with SU1498
or its solvent DMSO before being subjected to shear stress. Integrin
activation was assessed by immunoblotting to detect integrin-Shc
association and immunostaining to visualize integrin clustering. To
detect integrin-Shc association, the cells pretreated with SU1498 were
subjected to a shear stress of 12 dyn/cm2 for 30 min or
kept as static control. Cell lysates were immunoprecipitated with the
anti-
v
3 LM609 mAb, followed by
immunoblotting with the anti-Shc mAb. As shown in Fig.
6, the association of integrin
v
3 with Shc increased after shearing, and
SU1498 did not have a significant effect on this shear-induced action.
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DISCUSSION |
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Previous studies have shown that shear stress can activate both integrins (12) and Flk-1 (4). However, it remains unclear whether integrins and Flk-1 function in isolation or coordinate with each other in response to shear stress. Our results indicate an interplay between integrins and Flk-1 in response to shear stress. Whereas integrins play a critical role in mediating the shear-induced Flk-1 activation, Flk-1 is not essential for the shear-induced integrin activation. Therefore, our results indicate that integrins and Flk-1 play sequential roles in shear-induced signaling transduction.
Little is known about how cells perceive mechanical forces and convert
them into biochemical responses. Because many signal-transducing molecules concentrate in the focal adhesion complex that interconnects the cytoskeleton to integrins and ECM, it is very likely that integrins
and the focal adhesion complex may represent a major site of
integration of mechanotransduction processes (11). Indeed, it has been shown that integrin clustering induces the activation of
Flk-1 as well as other RTKs, including PDGFR and EGFR (22, 27,
39). Other reports have demonstrated that integrins serve as
potential mechanosensors for several shear-induced downstream signaling
pathways and that PDGF -receptor is tyrosine phosphorylated by
mechanical tension only when cells are plated on appropriate integrin
ligands (12, 39, 41). Our findings that inhibitory antibodies against
1- or
3-integrin
blocked the shear-induced Flk-1 · Cbl activation, although
noninhibitory antiserum against
1- or
3-integrin had no significant effects, provide evidence that the activation of integrins is essential for the shear activation of Flk-1 · Cbl. We hypothesize that the mechanically activated integrins, by assembling focal adhesion complexes and altering adhesion
forces, may reorganize the cytoskeletal network. This reorganization of
the cytoskeletal network may help to bring together different
molecules, including Flk-1 and Cbl, to a common signaling complex, thus
stabilizing the Flk-1 · Cbl association. That cytoskeleton may
play a critical role in transmitting signals from integrins to Flk-1 is
supported by the finding that cytochalasin D abolishes the
phosphorylation of PDGF-
-receptor in fibroblasts seeded on collagen
type I and fibronectin (39). Recent reports indicate that
1-integrin can associate with EGFR (24) and
that
3-integrin can bind with Flk-1 and PDGF
-receptor (2, 29). These associations between integrins
and RTKs may also take place via the cytoskeletal network, because
integrins themselves lack the capability of direct binding with
tyrosine kinases (31). In fact, both
v
3 and the activated PDGFR-
have been
found to be associated with a cytoskeletal (NP-40 insoluble) fraction
in stimulated cells (29). In addition, focal adhesion
complex contains a myriad of adapter and signaling molecules, including
Grb2 and Src, which have been documented to bind to both Flk-1 and Cbl.
Thus integrins may bring Flk-1 and Cbl to the proximity of focal
adhesion by recruiting adapter and signaling molecules such as Grb2 and Src.
Cbl plays an important role in tyrosine-phosphorylation-related signaling in cellular responses to extracellular cues. Many protein tyrosine kinases, including Src family tyrosine kinases and RTKs such as EGFR, PDGFR and CSF-1R, have been reported to associate with Cbl (21). Our results have shown that Cbl associates with Flk-1 in response to different types of stimuli, including shear stress, VEGF, and cell-ECM interaction (Figs. 1 and 2). Given that Cbl is composed of a number of functional motifs, including a phosphotyrosine-binding domain (PTB), a proline-rich region (PRO), and several tyrosine residues in an appropriate context to serve as potential binding sites for the SH2 domains of signaling molecules, it can be envisioned that Flk-1 · Cbl association can recruit different signaling molecules and activate multiple signaling pathways, thus amplifying the effects of Flk-1 activation. In fact, the disruption of Flk-1 · Cbl association by SU1498 (Fig. 5A) can significantly inhibit the shear-induced ERK activation (data not shown).
In the absence of stimulation, Grb2 constitutively associates with Flk-1 (13), and Cbl forms a complex with the SH3 domain of Grb2 via its PRO (21, 34). This provides a mechanism for Grb2 to bridge Cbl to Flk-1 without the involvement of tyrosine phosphorylation of Flk-1 and the PTB domain in Cbl. This hypothesis is supported by our findings on the nonstimulated cells in suspension that there is a significant association of Flk-1 with Cbl but no tyrosine phosphorylation of the Cbl-bound Flk-1 (Fig. 2). Both the association and the tyrosine phosphorylation of the Cbl-bound Flk-1 were substantially enhanced after stimulation by shear stress and adhesion, with the effect being stronger on tyrosine phosphorylation than on the association (Figs. 1A and 2). Therefore, in contrast to the lack of its involvement in nonstimulated cells, tyrosine phosphorylation appears to play a significant role in the Flk-1 · Cbl association upon stimulation, possibly via direct binding of the tyrosine-phosphorylated sites of Flk-1 to the PTB domain of Cbl. SU1498, which inhibits the tyrosine phosphorylation of Flk-1, blocked the shear-enhanced Flk-1 · Cbl association without reducing the basal association in the static controls (Fig. 5). Therefore, we postulate that the Flk-1 · Cbl association occurs via two different mechanisms. Under static condition, Cbl constitutively associates with Flk-1 through mediation by a third partner, such as the adapter molecule Grb2; upon stimulation, in addition to the constitutive Flk-1 · Cbl association, the PTB domain of Cbl may directly bind to the tyrosine phosphorylated sites on Flk-1 and thereby associate with the phosphorylated subfraction of Flk-1.
Soldi et al. (35) reported that endogenous
3 integrin is involved in the VEGF-induced Flk-1 and its
downstream PI3K activations in HUVEC. Borges et al. (2)
reported that recombinant
3, but not
1,
integrin is constitutively associated with Flk-1 in CHO cells. In our
study on BAECs, however, the ligation of
1, as well as
3, integrin caused a significant Flk-1 · Cbl
activation, and they are essential for the shear-induced
Flk-1 · Cbl activation (Figs. 2 and 3). There is evidence that
crosslinking or ligation of
1 integrin can cause the
translocation of Cbl to the membrane (18, 25, 28), which
may facilitate the Flk-1 · Cbl association.
In summary, our findings demonstrate that shear stress activates
integrins to transactivate Flk-1 and Cbl. Although Flk-1 is essential
for the activation of integrins in response to chemical stimulation
(VEGF) (3), our results on mechanotransduction demonstrate
that integrins are upstream to Flk-1 and that Flk-1 is not essential
for integrin activation (Fig. 8). These
results indicate that 1) different membrane receptors do not
function in isolation in mechanotransduction, and 2)
mechanical and chemical stimuli may regulate intracellular signaling
pathways via differing mechanisms.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Research Grants HL-19454, HL-43026, and HL-62747 (S. Chien) and a grant from the Cho Chang Tsung Education Foundation.
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FOOTNOTES |
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Present addresses: S. Li, Dept. of Biomedical Engineering, Univ. of California, Berkeley, Berkeley, CA; J.Y.-J. Shyy, Division of Biomedical Sciences, Univ. of California, Riverside, Riverside, CA.
Address for reprint requests and other correspondence: S. Chien, Dept. of Bioengineering, Univ. of California, San Diego, La Jolla, CA 92093-0427 (E-mail: shuchien{at}ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 24, 2002;10.1152/ajpcell.00222.2002
Received 16 May 2002; accepted in final form 17 July 2002.
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