Interplay between integrins and FLK-1 in shear stress-induced signaling

Yingxiao Wang, Hui Miao, Song Li, Kuang-Den Chen, Yi-Shuan Li, Suli Yuan, John Y.-J. Shyy, and Shu Chien

Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California at San Diego, La Jolla, California 92093


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 3- or beta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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 alpha vbeta 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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha vbeta 3 (LM609), anti-beta 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-alpha vbeta 3 LM609 mAb or the anti-beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Shear stress and VEGF induce Flk-1 · Casitas B-lineage lymphoma (Cbl) activation. Bovine aortic endothelial cells (BAECs) were either subjected to a shear stress of 12 dyn/cm2 (left) or treated with VEGF at 10 ng/ml (right) for various lengths of time as indicated. Cell lysates from the various samples were subjected to immunoprecipitation (IP) with an anti-Cbl antibody, followed by immunoblotting (IB) with an anti-Flk-1 antibody (top, arrow Flk-1), a PY20 anti-phosphotyrosine antibody (center, arrow P-Flk-1), or an anti-Cbl antibody (bottom, arrow Cbl). Bar graphs are densitometry analysis representing the means ± SE from 3 separate experiments. Asterisks indicate significant differences (P < 0.05) between sheared (left) or VEGF-treated (right) samples and their respective controls.

Chemical stimulation by VEGF for 5 min also induced both Flk-1 · Cbl association and Cbl-bound Flk-1 phosphorylation (Fig. 1, right). Although the induction of Flk-1 phosphorylation by VEGF is well established, this is the first demonstration that VEGF induces Flk-1 · Cbl association. Thus both mechanical (shear stress) and chemical (VEGF) stimuli can induce Flk-1 · Cbl activation.

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 alpha vbeta 3) or fibronectin (ligand for both alpha 5beta 1 and alpha vbeta 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-beta 1 or alpha vbeta 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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Fig. 2.   Integrin-ligand interaction induces Flk-1 · Cbl activation. BAECs were trypsinized and kept in suspension in 1% agarose gel-coated dishes for 60 min at 37°C. Cells were allowed to adhere to dishes coated with vitronectin (left) or fibronectin (right) for various periods of time as indicated. The procedures for IP and IB and arrows are the same as those described in Fig. 1. Shown in bar graphs in top and center are densitometry analysis representing the means ± SE from 3 separate experiments. Asterisks indicate significant differences (P < 0.05) between the various samples and their corresponding controls.

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 alpha vbeta 3- and beta 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 beta 3- or beta 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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Fig. 3.   Integrin integrity is essential for the Flk-1 · Cbl activation induced by shear stress. BAECs were treated with mouse IgG (10 µg/ml), LM609 (10 µg/ml), or 6S6 (10 µg/ml) for 2 h. The treated cells were subjected to shear stress or static incubation for 5 min. The procedures for IP and IB and the graphic presentations are the same as in Fig. 1. Bar graphs, representing the means ± SE from 3 separate experiments, show the band intensities of the various samples relative to those in the untreated, static controls. Asterisks indicate significant differences (P < 0.05) between the various sheared samples and their corresponding static controls.



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Fig. 4.   Integrin integrity is essential for the shear-induced tyrosine phosphorylation of Cbl. BAECs were treated with mouse IgG (10 µg/ml), LM609 (10 µg/ml), or 6S6 (10 µg/ml) for 2 h before being subjected to shear stress or static incubation for 5 min. Cell lysates were subjected to IP with an anti-Cbl antibody and IB with a PY20 anti-phosphotyrosine MAb (top) or an anti-Cbl antibody (bottom); arrows point to the tyrosine phosphorylated Cbl (P-Cbl) at top and total Cbl protein level at bottom. Graphic presentations are the same as in Fig. 1. #Significant differences (P < 0.05) between mouse IgG and LM609 or 6S6 treated cells after shearing.

We also examined the effects of integrins on the VEGF-induced Flk-1 · Cbl activation. BAECs were incubated with LM609 or mouse IgG before they were subjected to VEGF treatment. LM609 did not have any significant effect on the Flk-1 · Cbl activation induced by VEGF (data not shown), indicating that the Flk-1 · Cbl activation induced by VEGF is independent of integrins. Thus mechanical and chemical stimuli may induce Flk-1 · Cbl activation through different mechanisms.

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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Fig. 5.   Flk-1 is important for Flk-1 · Cbl association and tyrosine phosphorylation of Cbl. BAECs were treated with DMSO (0.1%) or SU1498 (5 µM) for 1 h before being subjected to shear stress or static incubation for 5 min. Cell lysates were subjected to IP with an anti-Cbl antibody and IB with an anti-Flk-1 antibody (top in A; arrow Flk-1), a PY20 anti-phosphotyrosine MAb (top in B, arrow P-Cbl), or an anti-Cbl antibody (bottom in A and B, arrows Cbl). Graphic presentations are the same as in Fig. 1. Asterisks indicate significant differences (P < 0.05) between the various sheared samples and their corresponding static controls.

Flk-1 is not essential for the integrin-Shc association and integrin clustering induced by shear stress. Because integrin beta 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-alpha vbeta 3 LM609 mAb, followed by immunoblotting with the anti-Shc mAb. As shown in Fig. 6, the association of integrin alpha vbeta 3 with Shc increased after shearing, and SU1498 did not have a significant effect on this shear-induced action.


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Fig. 6.   Flk-1 is not essential for the integrin-Shc association induced by shear stress. BAECs were treated with DMSO (0.1%) or SU1498 (5 µM) for 1 h before being subjected to shear stress or static incubation for 30 min. Cell lysates were subjected to IP with the anti-alpha vbeta 3 LM609 MAb and IB with an anti-Shc MAb. Arrows point to different isoforms of Shc coimmunoprecipitated with alpha vbeta 3-integrin. Bar graphs, representing the means ± SE from 3 separate experiments, show the band intensities of the various samples relative to those in the untreated, static controls. Asterisks indicate significant differences (P < 0.05) between various samples and their corresponding static controls.

Although alpha vbeta 3 was relatively dispersed in BAECs under static condition (Fig. 7Ai), it showed enhanced clustering on the abluminal side after shear stress (Fig. 7Aii). Quantification of the images (data not shown) revealed an increase in the size of the alpha vbeta 3 spots after shearing. Inhibition of Flk-1 with SU1498 did not have any significant effect on this shear-induced alpha vbeta 3 clustering (Fig. 7A, iii and iv). The same approach was applied to study beta 1 integrin. Whereas the clustering of alpha vbeta 3 forms spots (Fig. 7A, ii and iv), the clustering of beta 1-integrin forms thinner stripes (Fig. 7B, ii and iv). The inhibition of Flk-1 again did not affect the shear-induced clustering of beta 1-integrin (Fig. 7B, iii and iv).


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Fig. 7.   Flk-1 is not essential for the integrin clustering induced by shear stress. BAECs were pretreated with 0.1% DMSO (Fig 7A, i and ii; 7B, i and ii) or 5 µM SU1498 (Fig 7A, iii and iv; 7B, iii and iv) for 1 h before being subjected to shear stress (Fig 7A, ii and iv; 7B, ii and iv), with the direction of flow from left to right, or static incubation (Fig 7A, i and iii; 7B, i and iii) for 15 min. The cells were then fixed with methanol and immunostained with the anti-alpha vbeta 3 (A) or anti-beta 1 (B) MAbs together with Hoechst-33258. The images were collected by confocal microscopy, and the Adobe Photoshop was used to superimpose the staining of integrins (green) with the Hoechst-33258 staining of nuclei (red).

All these results on integrin-Shc association and integrin-clustering indicate that Flk-1 is not essential for the shear-induced activation of integrins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta 1- or beta 3-integrin blocked the shear-induced Flk-1 · Cbl activation, although noninhibitory antiserum against beta 1- or beta 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-beta -receptor in fibroblasts seeded on collagen type I and fibronectin (39). Recent reports indicate that beta 1-integrin can associate with EGFR (24) and that beta 3-integrin can bind with Flk-1 and PDGF beta -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 alpha vbeta 3 and the activated PDGFR-beta 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 beta 3 integrin is involved in the VEGF-induced Flk-1 and its downstream PI3K activations in HUVEC. Borges et al. (2) reported that recombinant beta 3, but not beta 1, integrin is constitutively associated with Flk-1 in CHO cells. In our study on BAECs, however, the ligation of beta 1, as well as beta 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 beta 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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Fig. 8.   A proposed model showing the interrelations of integrins and Flk-1 · Cbl in response to shear stress. Shear stress activates integrins to transactivate Flk-1, which subsequently binds to Cbl translocated from cytoplasm to the membrane. This association between Flk-1 and Cbl in turn induces the tyrosine phosphorylation of Cbl and creates potential docking sites for downstream signaling molecules.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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