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Integrins as key constituents of focal adhesions
Integrins are involved in many cellular processes, including migration, proliferation and cell survival (Urbich et al., 2000; Eliceiri, 2001; Urbich et al., 2002). They are heterodimeric proteins of the plasma membrane constituted by 
and 
subunits. There are eighteen or more different a subunits and at least eight subunits. Though not all possible combinations of subunits exist in vivo, at least twenty four different integrins are known at present (Hass et al., 2003; Bouvard et al., 2001). Both and subunits have a large extracellular portion, only one transmembrane domain and a short intracellular domain. One exception to this rule is 4 , which has a cytoplasmic domain more than one thousand aminoacids long (Ylanne, 1998).
A key feature of integrins is that they link extracelullar matrix proteins to cytoskeletal actin microfilaments, as well as intracellular signaling molecules involved in transduction. These associations are localized in discrete regions of the cell called focal adhesions. In these regions, many integrins cluster and contribute to attach the cell to its extracellular environment. Vinculin, paxilin, talin, tensin and -actinin are cytoskeletal proteins that link integrins and actin microfilaments. Cytoplasmic tails of integrins bind directly to talin and -actinin, which in turn bind to actin microfilaments and vinculin. The cytoplasmic domain of integrins is crucial for two types of integrin signaling: activation of intracellular signaling cascades after extracellular stimulation (outside-in signaling) and regulation of binding affinity by intracellular signals (inside-out signaling) (Schwartz et al., 1995; Clark and Brugge, 1995; Aplin et al., 1998; Boudreau and Jones, 1999).
Focal adhesions contain a number of enzymes and signaling molecules critical for integrin outside-in signaling. These molecules include tyrosine kinases, such as focal adhesion kinase (FAK), Fyn, Syk, Csk and c-Src. Other kinases such as integrin-linked kinase (ILK), PI-3K and PKC, adapter proteins such as Grb2, Shc, Crk, Nck and p130CAS, as well as other molecules such as ICAP1, SOS, C3G, caveolin, calreticulin, integrin-associated protein and GTPase regulator associated with FAK (GRAF) are also present. Interactions and intracellular signaling mediated by these molecules are complex and not yet completely understood, and a description of them is beyond the scope of this review (see for review Schwartz et al., 1995; Clark and Brugge, 1995; Aplin et al., 1998; Giancotti and Ruoslahti, 1999; Boudreau and Jones, 1999; Schwartz, 2001; Eliceiri, 2001).
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Focal adhesions are probably important sites for
integrin-dependent mechanotransduction
The cytoskeleton, a complex intracellular network composed of microtubules, actin microfilaments and intermediate filaments, confers shape and motility to cells. The actin microfilament network links cell structures such as plasma membrane, nuclear membrane, intercellular junctions and focal adhesions and transmits internally self-generated tension to these structures (Maniotis et al., 1997). In the opposite direction, external forces applied locally are distributed throughout the whole cell by the cytoskeletal network (Wang et al., 1993).
Mechanotransduction in adherent cells depends on force transmission to the transducer structures, which may be far away from the stimulation site. Integrin-containing focal adhesions are intensively studied regions where mechanotransduction probably takes place (Riveline et al., 2001; Katsumi et al., 2004). Davies et al. (1994) showed that shear stress at the luminal surface of endothelial cells in culture, biases remodeling of focal adhesions in the abluminal surface in the direction of flow, within a few minutes of constant exposure. This result suggests that the response of the cells to shear stress applied on the luminal surface implicates distribution of forces over the whole cytoskeletal network, which may affect abluminal focal adhesions, the major sites of integrin-mediated transduction (Davies and Barbee, 1994; Wozniak et al., 2004). Bundles of actin known as stress fibers interconnect focal adhesions, acting as force transmitters to these regions.
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Integrins and shear stress transduction
in vascular endothelial cells
- Signaling molecules involved in integrin-dependent transduction
Vascular endothelial cells are continuously exposed to shear stress associated to blood flow. These shearing forces have paramount importance in regulation of endothelial cell physiology in normal and pathological states (for reviews, Davies and Tripathi, 1993; Davies, 1994; Berthiaume and Frangos, 1994; Davies et al., 1997; Traub and Berk, 1998; Lehoux and Tedgui, 1998).
Integrins are present in the luminal and abluminal faces of endothelial cells, but they are more abundant in the abluminal side (Muller et al., 1997; Jalali et al., 2001; Tzima et al., 2001). Several integrins have been reported in endothelial cells, 
, 
, 
, 
, 
, 
, 
, 
, 
, 
and 
(Bouvard et al., 2001).
 |
Ishida et al. (1996) showed that shear stress applied to cultured endothelial cells from human umbilical vein (HUVECs) induces tyrosine phophorylation of several proteins, including FAK. This enzyme autophosphorylates when it is activated by integrins, allowing binding of other signaling molecules of the focal adhesion and leading to a signaling cascade (Schwartz et al., 1995; Aplin et al., 1998; Giancotti and Ruoslahti, 1999). Additionally, it was shown that FAK tyrosine phophorylation could also be achieved by using an antibody (8A2) that selectively activates  integrins. Both 8A2 and shear stress activated the MAP kinase (MAPK) pathway (Ishida et al., 1996). |
Li et al. (1997) reported that FAK activation by shear stress is dependent on integrin stimulation, since an anti- blocking antibody greatly attenuated it. In addition, the shear stress-induced integrin-dependent activation of FAK is impaired by disruption of actin microfilaments with cytochalasin (Li et al., 1997), which suggests that the shearing forces are transmitted by these microfilaments to focal adhesions, where the activation of FAK takes place. A major integrin-dependent MAPK activation pathway by shear stress in endothelial cells seems to be the FAK-Grb2-SOS-Ras pathway (Li et al., 1996, 1997).
Other signaling molecules also play important roles in integrin-dependent shear stress transduction. For example, Chen et al. (1999) reported that shear stress evokes an increased association between integrins and the adapter protein Shc, present in focal adhesions. Increased association with Shc was demonstrated for and integrins containing and 
subunits. These results suggest that several integrins may be involved in shear stress transduction, perhaps mediating different cellular responses to stress. Shear stress was also shown to induce increased tyrosine phosphorylation of Shc and Shc binding to Grb2, the latter suggesting a MAPK activation pathway. To test this hypothesis, endothelial cells were transfected with a dominant negative mutant of Shc, which resulted in great attenuation of MAPK activation by shear stress. Accordingly, this negative mutant also inhibited the shear stress-induced increase in the expression of genes regulated by the MAPK signaling pathway.
Src family kinases (SFKs) have also an important role in shear stress transduction by integrins. Shear stress activates SFKs in endothelial cells (Takahashi and Berk, 1996), resulting in activation of a p130 cas-Crk signaling pathway (Okuda et al., 1999) and caveolin-1 phosphorylation. This phosphorylation mediates Csk association at focal adhesions, where it plays a role in integrin-dependent myosin light chain phosphorylation and actin cytoskeleton reorganization triggered by shear stress (Radel and Rizzo, 2005). The small GTPases Rho, Rac and Cdc42, which play an important role in cytoskeleton organization, cell spreading and migration, are regulated by shear stress with relevant physiological consequences. Cytoskeletal alignment in the direction of flow, a key adaptation of endothelial cells under shear stress, depends on integrin-induced transient inactivation of Rho (Tzima et al., 2001). On the other hand, Rac is activated by shear stress downstream of integrin activation and mediates cytoskeletal reorganization and regulation of gene expression (Tzima et al., 2002). Shear stress-induced integrin-dependent activation of Cdc42 determines the positioning of the microtubule organizing center on the downstream side of the nucleus relative to the direction of flow (Tzima et al., 2003).
- Mechanism of integrin response to shear stress in endothelial cells
Jalali et al. (2001) introduced an interesting concept about integrin-dependent transduction of shear stress. First, they showed that shear stress increases integrin binding to their specific extracellular matrix ligands. This result suggested that ligand binding by integrins might be important in shear stress transduction. To assess this possibility, the authors studied the influence of different extracellular matrix proteins on the shear stress-induced association between integrins and Shc. Interestingly, they observed that this association occurred only when the integrin could bind to its specific ligand. For example, the laminin receptor associated to Shc only when endothelial cells were cultured on laminin, but not on vitronectin, fibronectin or collagen.
The constant remodeling of focal adhesions on the abluminal side of endothelial cells (Davies et al.,1994) suggests that dynamic integrin-ligand associations may be essential for mechanotransduction. In order to test this hypothesis, Jalali et al. (2001) blocked unoccupied (not bound to integrins) extracellular matrix proteins with the appropriate antibodies to prevent formation of new integrin-ligand binding in response to shear stress. The results of these experiments suggested that dynamic formation of new integrin-ligand associations is essential for integrin transduction of shear stress, since blockade of the unoccupied binding sites for integrin resulted in no significant increase in -Shc association when the cells were exposed to shear stress. Similar results were obtained targeting integrin .
These results indicate that mechanotransduction in endothelial cells in response to shear stress requires binding of integrins to their specific ligands and dynamic formation of new integrin-ligand associations.
To investigate whether shear stress affected the affinity of integrin binding to their ligands, Tzima et al. (2001) used WOW-1, a Fab fragment that selectively binds to when this integrin displays high affinity for vitronectin. When endothelial cells were stimulated with flow, there was no significant increase in total expression compared to control (non-sheared) cells, but WOW-1 labeling dramatically increased. This indicated that the overall increase in integrin-ligand binding was due to conformational changes induced by shear stress that shift integrins to a high affinity state. The same mechanism has been recently reported in NIH3T3 cells exposed to stretch (Katsumi et al., 2005).
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Approaches to investigate integrin-dependent mechanotransduction
Basically two strategies have been frequently used to study the involvement of integrins in mechanotransduction. The first one consists in antagonization of integrin signaling by the use of blocking antibodies or RGD peptides (MacKenna et al., 1998; Rainger et al., 1999; Liang et al., 2000). The second approach is to perform a specific mechanical stimulation, by directly twisting integrins through ferromagnetic microbeads coated with integrin ligands (Glogauer et al., 1995; Glogauer et al., 1997; Chicurel et al., 1998; Schmidt et al., 1998; Chen et al., 2001). Both strategies have been successful providing evidence of the important role of integrins in force tranduction in different cell types. These cell types include ventricular myocites (Liang et al., 2000), tracheal smooth muscle cells (Tang et al., 1999), vascular smooth muscle cells (Wilson et al., 1995; Davis et al., 2001), neutrophils (Rainger et al., 1999), fibroblasts (Glogauer et al., 1995; Glogauer et al., 1997; MacKenna et al., 1998), chondrocytes (Wright et al., 1996; Wright et al., 1997), osteoblast-like cells (Salter et al., 1997; Schmidt et al., 1998; Pavalko et al., 1998) and endothelial cells (see section 3 of this report).
.
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Role of 
integrin in flow transduction:
a novel approach
- Why the need for a novel approach?
As exposed along this article, a considerable group of evidences points to integrins as key players of shear stress transduction by endothelial cells. Since there are many different integrins, an important question is whether they play different roles in this particular form of transduction or not.
| Integrin has been positively identified as a shear stress sensor in endothelial cells (Li et al., 1997; Bhullar et al., 1998; Jalali et al., 2001; Tzima et al., 2001; Gloe et al., 2002; Wang et al., 2002), but other integrins also seem to play additional roles in flow sensing. Two distinctive effects of shear stress on endothelial cells, i.e. activation of the transcription factor SREBP1 (Liu et al., 2002) and induction of cell migration (Urbich et al., 2002), have been shown to be dependent on integrins. Probably, the different integrins do not have identical functions, and stimulation of one specific type can potentially give rise to cellular responses different from those obtained by stimulation of others (Leavesley et al., 1993; Schwartz et al., 1995). |
|
Integrin is a vitronectin receptor, just like . From studies in different cell types, such as osteoblasts, cardiac myocites and vascular smooth muscle cells, there is evidence that plays a role in mechanotransduction (Wilson et al., 1995; Salter et al., 1997; Liang et al., 2000). However, there is scarce evidence for α Vβ 5 functioning as a flow sensor in endothelial cells. In this regard, it is known that flow stimulation promotes the association between and the signaling molecule Shc in cultured endothelial cells, strongly suggesting that some of the effects of shear stress on these cells are due to activation of a mechanotransduction pathway involving and Shc (Chen et al., 1999). In addition, seems to have an important flow-sensing role in endothelial cells of coronary vessels, because an antibody directed against this protein is able to affect the dromotropic effect of coronary flow (Rubio and Ceballos, 2000).
Since endothelial cells express several different kinds of integrins and other proteins involved in flow sensing (see Fisher et al., 2001, for a review), it is difficult to attribute particular flow-induced responses to specific proteins. Therefore, we decided to test the sensitivity of to flow stimulation in a heterologous expression system, Xenopus laevis oocytes. The medullar hypothesis of this work was that shear stress stimulation of -expressing oocytes would result in the activation of endogenous oocyte ionic currents. Xenopus oocytes have the advantage to be, from a macroscopic point of view, electrophysiologically unresponsive to mechanical stimuli, including flow (Zhang and Hamill, 2000). Thus, any electrophysiological response to shear stress could be readily attributable to the expression of and not to an endogenous sensor. If was able to act as a flow sensor in the oocytes and could associate with signaling molecules that could lead to electrophysiological responses, then flow stimulation should result in activation of macroscopic currents, which can be easily recorded with standard techniques. Therefore, our experiments were designed to test the possible flow-sensing role of expressed in Xenopus oocytes using electrophysiological techniques.
In this kind of experiments, it is necessary to count with a positive control, in order to be sure that the experimental conditions are adequate to observe the expected responses. Inwardly rectifying potassium channels Kir2.1 are expressed in endothelial cells (Forsyth et al., 1997) and are activated in response to flow stimulation (Olesen et al., 1988). Furthermore, when Kir2.1 channels are expressed in Xenopus oocytes and these cells are stimulated with flow, electrophysiological responses resembling those of endothelial cells can be observed (Hoger et al., 2002). Therefore, we used Kir2.1-expressing oocytes as a positive control in our experiments.
Results and discussion
We verified that oocytes injected with the respective mRNAs of human or murine Kir2.1 actually expressed the corresponding proteins. An immunodetection method was applied in the case of . As shown in Fig. 1A, oocytes injected with mRNAs displayed positive immunoreactivity, while control oocytes did not. We did not observe any significant difference between the electrophysiological properties of -expressing and water-injected oocytes (data not shown). In the case of Kir2.1 mRNA-injected oocytes, we considered unnecessary to apply the immunodetection method, since we verified the expression of this channel by recording its characteristic currents.
Fig. 1
Fig. 1.Xenopus oocytes expressing a V b 5 are insensitive to flow stimulation. Manually defolliculated, stage V-VI Xenopus laevis oocytes were used for expression of human α V β 5 integrin and murine Kir2.1 channel. All cDNAs were transcribed in vitro with a commercial kit (m-MESSAGE-m-MACHINE, Ambion, Dallas, TX, USA). A day after defolliculation and at least 24 hrs. before recording, oocytes were pressure-injected with aqueous solutions of α V and β 5 or Kir2.1 mRNAs (23 ng of each mRNA). In all experiments, control oocytes were injected with equivalent amounts of water and tested in parallel with mRNA-injected oocytes. A. Immunodetection of α V β 5 . Lysates from 18 oocytes were prepared, and loaded onto nitrocellulose membranes. Membranes were incubated with mouse anti- α V β 5 primary antibody and horse-radish peroxidase-conjugated goat anti-mouse secondary antibody. Immunoreactivity was detected with ECL TM Chemiluminescence Western Blotting Detection Reagent (Amersham, Piscataway, NJ, USA). Only oocytes injected with α V β 5 mRNAs showed positive immunoreactivity. B. Top and side views of the basic experimental setup used. Macroscopic currents were recorded using the two-electrode voltage-clamp (TEVC) technique. Oocytes were placed in a small hole in the center of a recording chamber 20 mm long and 5 mm wide. In the scheme above, TEVC electrodes and glass pipette for flow stimulation are shown. Flow stimuli of varying rates (5-27 ml/min) were applied through the pipette for about 10 s. Shear stress was calculated using Stokes law, assuming that the oocyte is a sphere with a diameter of 1.3 mm. C. Flow stimulation of α V β 5 -expressing and control oocytes. Representative traces of α V β 5 -expressing (N=20), water-injected (N=12) and Kir2.1-expressing (N=43) oocytes are shown from left to right. Oocytes were clamped at –60 mV. The bars represent the time of flow stimulation. Below each bar, the stimulus intensity in ml/min and the corresponding shear stress value are indicated. Recording solution ND38 contained (in mM): 38 NaCl, 60 KCl, 1.8 CaCl 2, 1 MgCl 2, 5 HEPES, pH=7.5.
Then we explored the effect of flow stimulation in cells injected with Kir2.1 or mRNAs, or just water. We used flow rates ranging from 5 to 27 ml/min (corresponding shear stress values ranging from 1.4 to 7.7 dynes/cm 2). A general view of our stimulating-recording system is shown in Fig. 1B. Neither -expressing nor water-injected oocytes showed an electrophysiological response to flow stimulation at all potentials tested. In contrast, all cells expressing Kir2.1 responded with a slowly activating current, whose amplitude remained stable during the stimulus (Fig. 1C). We further characterized this shear stress-activated current. It showed strong inward rectification, since no outward current was observed for membrane values more positive than -20 mV (Fig. 2A). Current amplitude increased linearly with shear stress intensity in the range from 1.4 to 7.7 dynes/cm 2 (Fig. 2B) and it was abolished by 1 mM Ba 2+ (Fig. 2C).
FIG. 2
Fig. 2. Characterization of flow-induced responses in Kir2.1-expressing oocytes. A. Current-voltage relationship of theflow-activated current. From a holding potential of –20 mV, voltage steps from –160 to +60 mV lasting 1 s were presented in 20 mV increments. This protocol was applied in the absence and presence of flow stimulation (20 ml/min; 5.6 dynes/cm 2). At each voltage the currents in both conditions were subtracted from each other in order to obtain the current activated by flow (N=4). B. Amplitude of the flow-activated current as a function of stimulus intensity (N=5). Holding potential was –60 mV. C. Effect of 1 mM Ba 2+. In control conditions, this representative cell (N=5) showed a flow-induced response, which was abolished by addition of 1 mM Ba 2+. The bar represents the duration of flow stimulus (20 ml/min).
Does the lack of response to flow stimulation of -expressing oocytes rule out a role for this integrin as a flow sensor? Not necessarily, because alternative explanations may also account for this negative result. For example, this should be expected if does indeed act as a flow-sensing molecule in the oocytes, but the associated transduction mechanism is not coupled to activation of macroscopic currents. In this regard, even though no currents are activated, oocytes release ATP in response to flow stimulation, and this release is inhibited by RGD peptides (Maroto and Hamill, 2001). This suggests that oocytes indeed have a shear stress-induced signaling pathway that involves activation of endogenous integrins and produces a different outcome than ionic current activation. In the same way, it is possible that could activate signaling pathways not coupled to current activation. Other possible explanation is that is not able to act as a flow-sensing molecule in the oocytes because it requires specific conditions not present in these amphibian cells (e.g. binding to specific extracellular matrix proteins, interaction with signaling molecules, postranslational addition of specific glycosidic moieties) to carry out this function. In summary, our approach to elucidate a role for in shear stress transduction using Xenopus oocytes yielded a negative result, different explanations are possible and further investigation is necessary to test them.
Regarding the use of Kir2.1-expressing oocytes as positive controls in our experiments, we observed that it was appropriate. Identical flow stimuli than those applied on -expressing oocytes caused current activation in Kir2.1-expressing cells, indicating that the stimulation was adequate to activate flow-sensitive molecules. Thus, the negative result obtained in the case of cannot be explained by an inadequate stimulation system. The characteristics of the shear stress-activated current (strong inward rectification and Ba 2+ blockade) are indicative of Kir2.1 channel opening, and these results are consistent with those previously reported by Hoger et al. (2002). Our data basically reproduced their results, though the experimental conditions were slightly different. The Kir2.1 channel used in our experiments was from mouse macrophages, while that used by Hoger and collaborators was from bovine endothelial cells, and we found eight punctual differences between their submitted aminoacid sequences (GenBank accession numbers NM_008425 and NM_174373, respectively). Currently, the mechanism for the flow-induced activation of Kir2.1 channels is still unclear.
.
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Final remarks
There is strong evidence supporting an important role for integrins in shear stress transduction by endothelial cells. Focal adhesions on the abluminal surface of the cells are likely sites for this transduction to take place; actin microfilaments seem to be important in shearing force transmission to these sites. The transduction pathway is dependent on tyrosine kinase activation, since it is attenuated or abolished by tyrosine kinase inhibitors (Muller et al., 1996; Takahashi and Berk, 1996). Several proteins present in focal adhesions, e.g. FAK, Grb2, Shc, SOS and SFKs, have been involved in shear stress transduction by integrins. Integrin affinity for its ligands is increased by shear stress and dynamic formation of new integrin-ligand contacts seems to be essential for transduction. Integrin-mediated transduction of shear stress originates different endothelial cell responses such as basic fibroblast growth factor release (Gloe et al., 2002), cytoskeletal reorganization (Radel and Rizzo, 2005; Tzima et al., 2001,2002,2003), migration and cell motility (Urbich et al., 2002, Albuquerque and Flozak, 2003), changes in gene expression (Bhullar et al., 1998, Chen et al., 1999, Liu et al., 2002) and generation of anti-apoptotic signals (Urbich et al., 2000).
Finally, the expression of many different integrins in endothelial cells makes difficult the identification of specific roles for each one in transduction and final responses to flow stimulation. The approach described here tries to overcome this problem by studying individual integrins in an expression system, i.e. Xenopus oocytes. Albeit this time we obtained a negative result, we would like to remark that this fact does not discard the potential usefulness of this approach in future studies related to flow transduction.
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Acknowledgments
We thank Dr. John Noti (Guthrie Research Institute, Sayre, PA, USA) for his generous gift of α V and β 5 cDNAs. This work was supported by project G34998-N (CONACyT, Mexico), project PO1-HL18208 (National Heart, Lung and Blood Institute, NIH, USA), COPOCyT, Proyecto de Materiales Biomoleculares and Fondo de Apoyo a la Investigación ( San Luis Potosi, Mexico).
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Authors
Juan P. Reyes,
Instituto de Física, Universidad Autónoma de San Luis Potosí (UASLP); Ricardo Espinosa-Tanguma, Facultad de Medicina (UASLP); María A. Basurto, Facultad de Medicina (UASLP); Ulises Meza, Facultad de Medicina (UASP); Patricia Pérez-Cornejo, Facultad de Medicina (UASLP) y Center for Oral Biology, University of Rochester, NY, USA; Jorge Arreola, Instituto de Física (UASLP) y Center for Oral Biology, University of Rochester, NY, USA; Rafael Rubio, Facultad de Medicina (UASLP).
